ISOLATION AND CHARACTERIZATION OF BACTERIA AND FUNGI ASSOCIATED WITH FRESH FROZEN FISH

ISOLATION AND CHARACTERIZATION OF BACTERIA AND FUNGI ASSOCIATED WITH FRESH FROZEN FISH

ABSTRACT

Fish and fishery products have been documented as a major source of food borne pathogens of which bacteria and fungi play a major role. The fungal and bacterial organism contaminating apparently health and diseased (Scomber Scombrus) fish in Nasarawa, Nasarawa state was investigated. An invitro Assay was carried out to ascertain and identifies major bacterial and fungal contamination of frozen fish, which hitherto had constituted on important dieting intake of people of Nasarawa Nigeria. Fish samples collected were identified and the microbial load of the sample determined using spread plate method. Differentiation and characterization of various isolates were based on biochemical reactions and gram staining techniques. Frozen mackered fish (Scomber scombrus) was used for the study. The result revealed that staphylococcus aureus, Escherichia Coil, Salmonella Species and Shigella species are the bacteria associated with the contamination of the fish. Aspergillus, species Penicillin, species Rhizopus, species candida and mucor specie are the fungi associated with the contamination of the fish. The average number of colonies were 11, 21 and 45. The colony forming unit of O.5aiquote were 1.1 X i07, 2.1 x 106 and 4.5 x l0 isolation of fungi and Bacteria from Scomber Scombrus Meant for human consumption is alarming which can be source of fish borne infection. Attention should be paid to fish health in the community so as to conserve public health and safeguard the community health.

CHAPTER ONE

1.0       INTRODUCTION

Fish is one of the most important source of animal proteins available in the tropics and has been widely accepted as good source of protein and other elements for maintenance of healthy body (Al-jufalli and Opera, 2006). Fish is regarded as healthier meat due to its high content of long chain polysaturated fatty acids which are associated with improving health and preventing diseases of old age (yagoub & Ahmed, 2009).

The less developed countries capture 50% of the world harvest and a large proportion of the catch are consumed internally. In Nigeria, fish constitute 40% of the animal protein intake. Fish and fish products constitute an important part in the international trade, indicating increasing consumer interest in commodity. Generally, fish are good sources of vitamin B 12 and B6. It is also a good source of fluorine and iodine which are needed for the development of strong teeth and the prevention of goitre in man (youes & Bartram 2007). However, availability of these vital nutrients depends to a large extent on the methods of storage such as salting, roasting, drying, and freezing. Iced fish of different types are of great demands by the Nigerian consumers as a relatively cheaper source of animal protein. Fish and fish products are important, not only from the nutritional point of view but also as an item of international and foreign exchange carrier for a number of countries in the world (Yagoub, 2009).

Fish is a very perishable, high-protein food that typically contains a high level of free amino acids. Microbes rnetabolise these amino acids, producing ammonia, biogenic amines such as putrescine, histamine, cadaverine, organic acids, ketones and sulfur compounds (Emborg et al., 2005; Olatsdottir et al., 2005; Dalgard et al., 2006).

Microbiological quality is of importance to public health as it directly relates to spoilage of fish and becomes the cause of food poisoning. Microbial hazards causing infections and poor health are closely related to food safety concerned with animal proteins derived from marketed food- fish and fish products, meat and meat products. This creates a burning question for all consumers with a high risk commodity with regard to pathogenic bacteria & Fungi contaminations alarming to food safety challenge (Nilla et al., 2012). Refrigeration temperatures are also relevant because they are used by most households in Nigeria for temporary storage of fish (Oramadike et al., 201 0).Frozen state condition is also important since most fishes consumed in Nigeria are imported and usually come in frozen state (Oramadike et al., 2010).The quality of fish degrades due to a complete process in which physical, chemical and microbiological forms of deteriorations are implicated.
Therefore maintenance of quality is of utmost importance in the production and trade of fish and fish products. Although only a few infectious agents in fish are able to infect humans, some exceptions exists that may result in fatalities. However, the greatest risk to human health is due to the consumption of raw or insufficiently processed fish and fish products (Yagoub and Ahmed 2009).

According to the Centre For Food Safety And Applied Nutrition in Washington (2001),most fish related food borne illness are traced to Salmonella, Staphylococciisspp, Escherichia spp, Murcor spp penicillin spp, candida spp. Rhizopus and aspergillus. (Yagoub and Ahmed 2009). Spoilage bacteria differ somewhat for freshwater aid marine fish and for temperate and tropical water fish, storage and processing conditions also affect the predominant species on iced fish under aerobic conditions (Hozbor ci’ al., 2006). Packing under carbon dioxide and low concentration of sodium chloride favors the growth lactic acid bacteria and Photobacterium phosphoreuni. Microorganisms are found mostly on the skin, gills, operculum and intestines of life and newly caught fish. Fish contamination can be linked to raw material, personnel, processing tools such as forklifts through leakage, openings in buildings and pets. Some pathogens may even become established in the processing plants from niches where they can survive for a long period of time. The quality of our fish is of major concern to the food processors, consumers and public health authorities and provisions of safe, wholesome and acceptable fish and fish products to consumers and control of microorganisms is essential to meet these objectives.

The potentials of seafood to harbor microbial athogens and causing subsequent illness is well documented for both developed and developing countries (Younes and Bartram, 2007; Wright et cii., 2004). Water related diseases continue to be one of the major health problems globally (Adebayo et al., 2012). Okonko et al., (2009), reported that both bacteria and fungi are common flora of iced fish and fish related products during packaging. Majority of iced fish are sold in the open markets. Arannilewa et at., (2005) noted that protein decreased with increasing duration of frozen storage with fresh samples not frozen having higher protein content. Disadvantages such as product dehydration, rancidity, drip loss and product bleaching have an overall effect on the quality of frozen food. In spite of some disadvantages associated with frozen storage, freezing is .accepted as the effective way of preserving fish (Arannilewa e tal.,2005). The effect of physical conditions and chemical agents on the growth of microorganisms in fish has been investigated and well documented. In recent time, modern biotechnology have introduced new techniques that detect early fish contamination, improve taste, modify the quality of the fish, prolong the shelf life and also impact disease resistance to the fish Oramadike et a!., 2010).

Majority of the iced fish marketed in Nasarawa state are sold in open markets and so are exposed to dust and contaminated air, encouraging the growth of microorganisms which might facilitate increased transmission and prevalence of pathogens. Moreover poor personal hygiene, such as neglecting to wash hands after handling contaminated materials encourage contamination of iced fish with pathogenic organisms. This study is to assess the Microbial Load of fish sold at Nasarawa market in Nasarawa state. Also to isolate/identify the bacteria & fungi responsible for the contamination of frozen fish retailed at Nasarawa market

Bacteria
Bacteria are group of micro organism all of which lack a distinct nuclear membrane (hence are considered more primitive than animal and plant cells) and most of which have a cell wall of unique composition. Most bacteria are unicellular, the cells may be spherical (cocci) rod-shaped (bacillus) spiral  (spirillum) comma-shaped (vibrio) or cork screw shaped (Geirocheate). Generally they range in size between 0.5 and 5nlm.
Fungi
Fungi can be single celled or very complex multicellular organisms. They are found in just about any habitat but most live on the land mainly in soil or plant material rather than in sea or fresh water. A very small number of fungi cause disease in animals

1.1       Statement of Problem

Fish is mostly spoiled by bacteria and fungi microbiological quality is of importance to public health as it directly relates to spoilage of fish and becomes the cause of food poisoning. Microbial hazard causing infection and poor health are closely related to food safety concerned with animal protein delivered from marketed fish and fish product meat and meat product. This creates a burning question for all consumers with a high risk commodity with regards to pathogenic bacteria continuous alarming to food safety challenge. The isolation and characterization of these spoilage organism will go a long way to profer solution no. mater how little to this problem.

1.2       Justification
The study will help in the acquisition of the knowledge of fungi and bacteria associated or involved with the spoilage of fish, factors that enhance spoilage of fish and the risk involved in the consumption of spoiled fish.

1.3       Scope of The Study
The project work is limited to the isolation and characterization of bacteria and fungi associated with fresh fish sold around Nasarawa Local Government. This will help in creasing the health awareness of the public on the damages of eating uncooked or under cooked fish. The project work would have covered the whole of Nasarawa State but it was not possible due to financial and time constraint

1.4       Aim of The Study
To isolate and identify micro-organism associated with contamination of frozen fresh fish

1.5       Objective of The Study
The objective of the study are:

i. To determine the microbial Load of the Fish
ii. To identify the type of bacteria and fungi associated with frozen fish commonly consumed by people in this locality

Bacterial Host Specificity

Bacterial Host Specificity

The bacterial host range of phage is generally narrower than that found in the antibiotics thathave been selected for clinical applications. Mostphage are specific for one species of bacteria andmany are only able to lyse specific strains within a species. This limited host range can be advantageous, in principle, as phage therapy results in less harm to the normal body flora and ecology than commonly used antibiotics, which often disrupt the normal gastrointestinal flora and result in opportunistic secondary infections by organisms such as Clostridium difficile. The potential clinical disadvantages associated with the narrow host range of most phage strains is addressed through the development of a large collection of well-characterized phage for a broad range of pathogens, and methods to rapidly determine which of the phage strains in the collection will be effective for any given infection.

Advantages over Antibiotics

Phage therapy can be very effective in certainconditions and has some unique advantages overantibiotics. Bacteria also develop resistance tophages, but it is incomparably easier to developnew phage than new antibiotic. A few weeks versus years are needed to obtain new phage for new strain of resistant bacteria. As bacteria evolve resistance, the relevant phages naturally evolve alongside. When super bacterium appears, the super phage already attacks it. We just need to derive it from the same environment. Phages have special advantage for localized use,

Because they penetrate deeper as long as theinfection is present, rather than decrease rapidlyin concentration below the surface like antibiotics. The phages stop reproducing once as the specific bacteria they target are destroyed. Phages do not develop secondary resistance, which is quite often in antibiotics. With the increasing incidence of antibiotic resistant bacteria and a deficit in the development of new classes of antibiotics to counteract them, there is a need to apply phages in a range of infections.

Lytic phages are similar to antibiotics in thatthey have remarkable antibacterial activity. However, therapeutic phages have some advantages over antibiotics, and phages have been reported to be more effective than antibiotics in treating certain infections in humans and experimentally infected animals. For example, in one study, Staphylococcus aureus phages were used to treat patients having purulent disease of the lungs and pleura. The patients were divided into two groups; the patients in group A (223 individuals) received phages, and the patients in group B (117 individuals) received antibiotics. Also, this clinical trial is one of the few studies using phage administration (48 patients in group A received phages by injection). The results were evaluated based on the following criteria: general condition of the patients, X-ray examination, reduction of purulence, and Microbiological analysis of blood and sputum. No side effects were observed in any of the patients, including those who received phages intravenously. Overall, complete recovery was observed in 82% of the patients in the phage-treated group as opposed to 64% of the patients in the antibiotic-treated group. Interestingly, the percent recovery in the group receiving phages intravenously was even higher (95%) than the 82% recovery rate observed with all 223 phage-treated patients.

Therapeutic Use of Phages and Antibiotics

Antibiotics target both pathogenic microorganismsand normal micro flora. This affects the microbialbalance in the patient, which may lead to serioussecondary infections.

High specificity may be considered to be adisadvantage of phages because the disease-causing bacterium must be identified before phage therapy can be successfully initiated. Antibiotics have a higher probability of being effective than phages when the identity of the etiologic agent has not been determined. Replicate at the site of infection and are thus available where they are most needed.

They are metabolized and eliminated from thebody and do not necessarily concentrate at thesite of infection. The “exponential growth” of phages at the site of infection may require less frequent phage administration in order to achieve the optimal therapeutic effect. No serious side effects have been described. Multiple side effects, including intestinal disorders, allergies, and secondary infections (e.g., yeast infections) have been reported.

A few minor side effects reported fortherapeutic phages may have been due to the

liberation of endotoxins from bacteria lysed invivo by the phages. Such effects also may be observed when antibiotics are used. Phage-resistant bacteria remain susceptible to other phages having a similar target range.

Resistance to antibiotics is not limited totargeted bacteria. Because of their more broad-spectrum activity, antibiotics select for many resistant bacterial species, not just for resistant mutants of the targeted bacteria. Selecting new phages (e.g., against phage-resistant bacteria) is a relatively rapid process that can frequently be accomplished in days or weeks. Developing a new antibiotic (e.g., against antibiotic-resistant bacteria) is a time-consuming process and may take several years (16, 51).Evolutionary arguments support the idea thatactive phages can be selected against every antibiotic-resistant or phage-resistant bacterium bythe ever-ongoing process of natural selection.

(2)     PROTOPLASMIC FUSION

Somatic fusion, also called protoplastfusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between different varieties of the same species (e.g. between non-flowering potato plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale to produce Triticale).

Uses of somatic fusion include

Making potato plants resistant to potato leaf roll disease. Through somatic fusion, the crop potato plant Solanum tuberosum – the yield of which is severely reduced by a viral disease transmitted on by the aphid vector – is fused with the wild, non-tuber-bearing potato Solanum brevidens, which is resistant to the disease. The resulting hybrid has the chromosomes of both plants and is thus similar to polyploid plants. Somatic Hybridization was first introduced by Carlson in Nicotiana ‘glauea’ Process for plant cells

The somatic fusion process occurs in four steps:

·        The removal of the cell wall of one cell of each type of plant using cellulose enzyme to produce a somatic cell called a protoplast

·        The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei. The resulting fused nucleus is called heterokaryon.

·        The somatic hybrid cell then has its cell wall induced to form using hormones

·        The cells are then grown into calluses which then are further grown to plantlets and finally to a full plant, known as a somatic hybrid. Different from the procedure for seed plants describe above, fusion of moss protoplasts can be initiated without electric shock but by the use of polyethylene glycol (PEG). Further, moss protoplasts do not need phytohormones for regeneration , and they do not form a callus.

Instead, regenerating moss protoplasts behave likegerminating moss spores. Of further note sodium nitrate and calcium ion at high pH can be used, although results are variable depending on the organism.

Applications in animal cells

Somatic cells of different types can be fusedto obtain hybrid cells. Hybrid cells are useful ina variety of ways, e.g.,

Ø to study the control of cell division andgene expression,

Ø to investigate malignant transformations ,

Ø to obtain viral replication,

Ø for gene or chromosome mapping and for

Ø Production of monoclonal antibodies byproducing hybridoma (hybrid cells between an immortalized cell and an antibody producing lymphocyte), etc.

(3)     MITOCHONDRIAL INHERITANCE IN YEAST

Mitochondria are a central platform for diverse cellular functions including respiration, metabolite biosynthesis, ion homeostasis and apoptosis. Mitochondrial DNA (mtDNA), which encodes subunits of the oxidative phos-phorylation (OXPHOS) complexes, is pivotal for ensuring functional mitochondria and has an active role in determining phenotypic diversity and fitness in animals and budding yeasts owing to sophisticated interactions between the mitochondrial and chromosomal genomes.

Mitochondrial partitioning during mitosis in yeast (Saccharomyces cerevisiae).Extra-nuclear inheritance or cytoplasmic inheritance is the transmission of genes that occur outside the nucleus . It is found in most eukaryotes and is commonly known to occur in cytoplasmic organelles such as mitochondria and chloroplasts or from cellular parasites like viruses or bacteria.Mitochondria are organelles which function to produce energy as a result of cellular respiration. Chloroplasts are organelles which function to produce sugars via photosynthesis in plants and algae. The genes located in mitochondria and chloroplasts are very important for proper cellular function, yet the genomes replicate independently of the DNA located in the nucleus, which is typically arranged in chromosomes that only replicate one time preceding cellular division. The extranuclear genomes of mitochondria and chloroplasts however replicate independently of cell division. They replicate in response to a cell’s increasing energy needs which adjust during that cell’s lifespan. Since they replicate independently, genomic recombination of these genomes is rarely found in offspring contrary to nuclear genomes, in which recombination is common. Mitochondrial disease are received from the mother, sperm does not contribute for it.

Three general types of extranuclear inheritance exist.

·        Vegetative segregation results from random replication and partitioning of cytoplasmic organelles. It occurs with chloroplasts and mitochondria during mitotic cell divisions and results in daughter cells that contain a random sample of the parent cell’s organelles. An example of vegetative segregation is with mitochondria of asexually replicating yeast cells.

·        Uniparental inheritance occurs in extranuclear genes when only one parent contributes organellar DNA to the offspring. A classic example of uniparental gene transmission is the maternal inheritance of human mitochondria. The mother’s mitochondria are transmitted to the offspring at fertilization via the egg. The father’s mitochondrial genes are not transmitted to the offspring via the sperm. Very rare cases which require further investigation have been reported of paternal mitochondrial inheritance in humans, in which the father’s mitochondrial genome is found in offspring. Chloroplast genes can also inherit uniparentally during Sexual reproduction. They are historically thought to inherit maternally, but paternal inheritance in many species is increasingly being identified. Inheritance from species to species differs greatly and is quite complicated. For instance, chloroplasts have been found to exhibit maternal, paternal and biparental modes even within the same species.

·        Biparental inheritance occurs in extranuclear genes when both parents contribute organellar DNA to the offspring. It may be less common than uniparental extranuclear inheritance, and usually occurs in a permissible species only a fraction of the time. An example of biparental mitochondrial inheritance is in the yeast Saccharomyces cerevisiae. When two haploid cells of opposite mating type fuse they can both contribute mitochondria to the resulting diploid offspring.

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