Laser Induced Fluorescence Based Optical Fiber Probe for Analyzing Bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus smithii , and fibrin alginolvticus, a Gram- negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smithii was observed . The effect of dye on the samples was also studied in detail.

Laser induced fluorescence based optical fiber probe for analyzing bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus .cmithii , and fibrin alginolvticus, a Gram-' negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smitltii was observed . The effect of dye on the samples was also studied in detail.

Bioactivity Profile of Polyunsaturated Fattyacid extracts from Sardinella longiceps and Sardinella fimbriata – A comparative study

The oceans have proved to be an interminable source of new and effective drugs. Innumerable studies have proved that specific compounds isolated from marine organisms have great nutritional and pharmaceutical value. Polyunsaturated fattyacids (PUFA) in general are known for their dietary benefits in preventing and curing several critical ailments including Coronary heart disease (CHD) and cancers of various kinds. Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) are two PUFA which are entirely marine in origin – and small Clupeoid fishes like sardines are known to be excellent sources of these two compounds. In this study, we selected two widely available Sardine species in the west coast, Sardinella longiceps and Sardinella fimbriata, for a comparative analysis of their bioactive properties. Both these sardines are known to be rich in EPA and DHA, however considerable seasonal variation in its PUFA content was expected and these variations studied. An extraction procedure to isolate PUFA at high purity levels was identified and the extracts obtained thus were studied for anti-bacterial, anti-diabetic and anti-cancerous properties.Samples of both the sardines were collected from landing centre, measured and their gut content analysed in four different months of the year – viz. June, September, December and March. The fish samples were analyzed for fattyacid using FAME method using gas chromatography to identify the full range of fattyacids and their respective concentration in each of the samples. The fattyacids were expressed in mg/g meat and later converted to percentage values against total fatty acids and total PUFA content. Fattyacids during winter season (Dec-Mar) were found to be generally higher than spawning season (June-Sept). PUFA dominated the profiles of both species and average PUFA content was also higher during winter. However, it was found that S. longiceps had proportionately higher EPA as compared to S. fimbriata which was DHA rich. Percentage of EPA and DHA also varied across months for both species – the spawning season seemed to show higher EPA content in S. longiceps and higher DHA content in S. fimbriata. Gut content analysis indicate that adult S. fimbriata is partial to zooplanktons which are DHA rich while adult S. longiceps feed mainly on EPA rich phytoplankton. Juveniles of both species, found mainly in winter, had a gut content showing more mixed diet. This difference in the feeding pattern reflect clearly in their PUFA profile – adult S. longiceps, which dominate the catch during the spawn season, feeding mostly on phytoplankton is concentrated with EPA while the juveniles which are found mostly in the winter season has slightly less EPA proportion as compared to adults. The same is true for S. fimbriata adults that are caught mostly in the spawning season; being rich in DHA as they feed mainly on zooplankton while the juveniles caught during winter season has a relatively lower concentration of DHA in their total PUFA.Various extraction procedures are known to obtain PUFA from fish oil. However, most of them do not give high purity and do not use materials indicated as safe. PUFA extracts have to be edible and should not have harmful substances for applying on mice and human subjects. Some PUFA extraction procedures, though pure and non-toxic, might induce cis-trans conversions during the extraction process. This conversion destroys the benefits of PUFA and at times is harmful to human body. A method free from these limitations has been standardized for this study. Gas Chromatography was performed on the extracts thus made to ensure that it is substantially pure. EPA: DHA ratios for both samples were derived - for S. longiceps this ratio was 3:2, while it was 3:8 for S. fimbriata.Eight common strains of gram positive and gram negative bacterial strains were subjected to the PUFA extracts from both species dissolved in acetone solution using Agar Well Diffusion method. The activity was studied against an acetone control. At the end of incubation period, zones of inhibition were measured to estimate the activity. Minimum inhibitory concentration for each of the active combinations was calculated by keeping p < 0.01 as significant. Four of the bacteria including multi-resistant Staphylococcus aureus were shown to be inhibited by the fish extracts. It was also found that the extracts from S. fimbriata were better than the one from S. longiceps in annihilating harmful bacteria.Four groups of mice subjects were studied to evaluate the antidiabetic properties of the PUFA extracts. Three groups were induced diabetes by administration of alloxan tetra hydrate. One group without diabetes was kept as control and another with diabetes was kept as diabetic control. For two diabetic groups, a prescribed amount of fish extracts were fed from each of the extracts. The biochemical parameters like serum glucose, total cholesterol, LDL & HDL cholesterol, triglycerides, urea and creatinine were sampled from all four groups at regular intervals of 7 days for a period of 28 days. It was found that groups fed with fish extracts had marked improvement in the levels of total LDL & HDL cholesterol, triglycerides and creatinine. Groups fed with extracts from S. fimbriata seem to have fared better as compared to S. longiceps. However, both groups did not show any marked improvement in blood glucose levels or levels of urea.Cell lines of MCF-7 (Breast Cancer) and DU-145 (Prostate Cancer) were used to analyse the cytotoxicity of the PUFA extracts. Both cell lines were subjected to MTT Assay and later the plates were read using an ELISA reader at a wavelength of 570nm. It was found that both extracts had significant cytotoxic effects against both cell lines and a peak cytotoxicity of 85-90% was apparent. IC50 values were calculated from the graphs and it was found that S. longiceps extracts had a slightly lower IC50 value indicating that it is toxic even at a lower concentration as compared to extracts from S. fimbriata.This study summarizes the bioactivity profile of PUFA extracts and provides recommendation for dietary intake; fish based nutritional industry and indigenous pharmaceutical industry. Possible future directions of this study are also elaborated.

lncidence of enterotoxigenic Staphylococcus aureus in relation to the microbial safety of seafood

A detailed study was made on the microbial quality, with special reference to food safety, of the fish and fishery products in the retail trade in Cochin and around. Also, farmed molluscan shellfishes like mussels and oysters were investigated for the microbial quality including the presence of pathogenic bacteria. Special stress has been given to monitor the incidence of coagulase positive as well as coagulase negative Staphylococcus in these products and their relative incidence have been recorded.In the next part, the investigation was centered mainly on toxigenic S.aureus. This is because among the Gram positive toxigenic bacteria, the Saureus with potential to produce thermostable enterotoxins are more relavent in food safety conceming seafoods in comparison with the Gram-negative pathogens like Salmonella and V.cholerae.The incidence, toxigenic potential and conditions of toxin production by S.aureus have been investigated in detail. An attempt has also been made to relate the toxigenisis with the presence of the concerned toxigenic genes in the genomes of S. aureus strains.

Polypropylene/metal oxide nanocomposites: fiber spinning and evaluation”

PP has been getting much attention over the years because it is a very durable polymer commonly used in aggressive environments including automotive battery casings, fuel containers etc. They are used to make bottles, fibers for clothing, components in cars etc. However, it has some shortcomings such as low dimensional and thermal stability. Materials such as metal oxides with sizes of the order 1–50 nm have received a great deal of attention because of their versatile applications in polymer/ inorganic nanocomposites, optoelectronic devices, biomedical materials, and other areas. They are stable under harsh process conditions and also regarded as safe materials to human beings and animals. In the present investigation, PP is modified by incorporating metal oxide nanoparticles such as ZnO and TiO2 by simple melt mixing method. Melt spinning method was used to prepare PP/metal oxide nanocomposite fibers. Various studies have been carried out on these composites and fibers. In the first part of the study, ZnO nanoparticles were prepared from ZnCl2 and NaOH in presence of chitosan, PVA, ethanol and starch. This is a simple and inexpensive method compared to other methods. Change in morphology and particle size of ZnO were studied. Least particle size was obtained in chitosan medium. The particles were characterized by using XRD, SEM, TEM, TGA and EDAX. Antibacterial properties of ZnO prepared in chitosan medium (NZO) and commercial zinc oxide (CZO) were evaluated using a gram positive and a gram negative bacteria

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation.

Biochemical and physiological characteristics of actinomycetes isolated from high altitude shola soils of tropical Montane forest

Actinomycetes are gram-positive, free-living, saprophytic bacteria widely distributed in soil, water and colonizing plants showing marked chemical and morphological diversity. They are potential source of many bioactive compounds, which have diverse clinical effects and important applications in human medicine. In the present work, we have studied some of the physiological and biochemical characteristics of 36 actinomycete strains isolated from the shola soils of tropical montane forest; a relatively unexplored biodiversity hotspot. Ability of actinomycetes isolates to ferment and produce acids from various carbohydrate sources such as innositol, mannose, sorbitol, galactose, mannitol, xylose, rhamnose, arabinose, lactose and fructose were studied. Almost all the carbon compounds were utilized by one or other actinomycete isolates. The most preferred carbon sources were found to be xylose (94.44%) followed by fructose and mannose (91.66%). Only 41.76% of the isolates were able to ferment lactose. The ability of actinomycetes isolates to decompose protein and amino acid differ considerably. 72.22% of the isolates were able to decompose milk protein casein and 61.11% of the isolates decompose tyrosine. Only 8.33% of the strains were able to decompose amino acid hypoxanthine and none of them were able to decompose amino acid xanthine. Potential of the actinomycetes isolates to reduce esculin, urea and hippurate and to resist lysozyme was also checked. 91.66% of the isolates showed ability to decompose esculin and 63.88% of the isolates had the capacity to produce urease and to decompose urea. Only 25% of the isolate were able to decompose hippurate and 94.44% showed lysozyme resistance

Development of nitrifying bacterial consortia for immobilizing in nitrifying bioreactors designed for penaeid and non-penaeid larval rearing systems in the tropics

Two ammonia oxidizing (AMOPCU-1 and AMONPCU-1) and two nitrite oxidizing (NIOPCU-1 and NIONPCU-1) consortia for activating nitrifying bioreactors and thereby establishing nitrification in penaeid and non-penaeid hatchery systems were developed by enrichment. For further amplification of the consortia a simple medium having seawater (either salinity 30 ‰ or 15 ‰) as base, supplemented with NH4+-N/NO2--N and PO4- and pH adjusted to 8 was identified. During the amplification in a fermentor the consortia exhibited excessive wall growth and diminished their yield coefficient posing difficulty in harvesting the cells completely. The consortia consisted of both Gram negative and Gram-positive bacterial cells embedded in a mucilaginous matrix of glycocalyx - like material presumably composed of polysaccharides. The consortia besides being useful in activating nitrifying bioreactors developed for shrimp/prawn hatchery systems can also be used as bioaugmentors in the bioremediation of ammonia and nitrite toxicity in aquaculture systems.

Characterization of Bacteriocins from Bacillus licheniformis strain BTHT8 and Bacillus subtilis strain BTFK101 isolated from marine sediment

Pathogenic microorganisms such as Bacillus cereus, Listeria Monocytogenes and Staphylococcus sp have caused serious diseases, and consequently contributed to considerable economic loss in the food and agricultural industries. Antibiotics have been practically used to treat these pathogens since penicillin G was discovered more than half a century ago. Many different types of antibiotics have been discovered or synthesized to control pathogenic microorganisms. Repetitive use and misuse of antibiotics by the agricultural and pharmaceutical industries have caused the emergence of multidrug-resistant microorganisms, even to the strongest antibiotics currently available; therefore, the rapid development of more effective antimicrobial compounds is required to keep pace with demand. Bacteria were isolated from marine water and sediment samples collected from various locations off the coast of Cochin and salt pans of Tuticorin using pour plate technique. One hundred and twelve isolates were obtained. Seventeen isolates exhibiting antimicrobial activity were segregated after primary screening. The secondary screening which was aimed at selection of bacteria that produce proteinaceous inhibitory compounds, helped to select five strains viz. BTFK101, BTHT8, BTKM4, BTEK16 and BTSB22. The five isolates inhibited the growth of six Gram positive test organisms viz. B. cereus, B. circulans, B. coagulans, B. pumilus, Staphylococcus aureus and Clostridium perfringens. After quantitative estimation of the bacteriocin production, the two strains BTFK101 and BTHT8 were selected for further study.

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.