Studies on isolation and molecular characterization of plasmids from Vibrios

While the seriousness of the problem of antibiotic resistance is now recognized, the complex web of resistance linking humans, animals, and the environment is getting realized. More often, antibiotics are used as a preventive measure against diseases. Antibiotic use for agriculture leads to the increased resistance in the environment since antibiotics are inevitable element during agriculture/aquaculture and antibiotic residues are excreted as waste that is frequently spread onto farmland as organic fertilizer. Fecal bacteria survive long periods in the environment and spread through runoff into groundwater, rivers, and marine ecosystems.However, horizontal gene transfer occurs in the animals and guts of humans and in a variety of ecosystems, creating a pool of resistance in the rice fields and open waters. Even if people are not in direct contact with resistant disease through food animals, there are chances of contact with resistant fecal pathogens from the environment. Additionally, pathogens that are autochthonous to the environment can acquire resistance genes from the environment. Our study revealed that autochthonous , bacteria Vibrio spp gained antibiotic resistance in the environment. Further, it was evident that horizontal gene transfer occurs in Vibrio by means of plasmids, which further augments the gravity of the problem. Non-pathogenic bacteria may also acquire resistance genes and serve as a continuing source of resistance for other bacteria, both in the environment, and in the human gut. As the effectiveness of antibiotics for medical applications decline, the indiscriminate use of in aquaculture and in humans can have disastrous conditions in future due to horizontal gene transfer and the spread of resistant organisms: We must recognize and deal with the threat posed by overuse of antibiotics.

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.

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.

Distribution and bioactive potential of soil actinomycetes from different ecological habitats

Emergence of drug resistance among pathogenic bacteria to currently available antibiotics has intensified the search for novel bioactive compounds from unexplored habitats. In the present study actinomycetes were isolated from two relatively unexplored and widely differing habitats such as mountain and wetlands and their ability to produce antibacterial substances were analyzed. Pure cultures of actinomycetes were identified by morphological and biochemical tests. Various genera of actinomycetes encountered included Nocardia, Pseudonocardia, Streptomyces, Nocardiopsis, Streptosporangium, Micromonospora, Rhodococcus, Actinosynnema, Nocardiodes, Kitasatosporia, Gordona, Intrasporangium and Streptoalloteichus. The frequency of occurrence of each genus was found to vary with sample. About 47% of wetland isolates and 33% of mountain isolates were identified as various species of Nocardia. The isolated strains differed among themselves in their ability to decompose proteins and amino acids and also in enzyme production potential. Antibiotic activities of these actinomycetes were evaluated against 12 test pathogenic bacteria by well diffusion method using agar wells in glycerol-yeast extract agar. About 95% of actinomycete isolates from wetland ecosystem and 75% of highland isolates suppressed in different degrees the growth of test pathogens. Relatively high antibacterial activity among these isolates underlined their potential as a source of novel antibiotics.

“Effect of spice oleoresins in microbial decontamination and their potential application in quality stabilization of Tuna (Euthynnus affinis) during storage”

Unprocessed seafood harbor high number of bacteria, hence are more prone to spoilage. In this circumstance, the use of spice in fish for reduction of microorganism can play an important role in seafood processing. Many essential oils from herbs and spices are used widely in the food, health and personal care industries and are classified as GRAS (Generally regarded as safe) substances or are permitted food additives. A large number of these compounds have been the subject of extensive toxicological scrutiny. However, their principal function is to impart desirable flavours and aromas and not necessarily to act as antimicrobial agents. Given the high flavour and aroma impact to plant essential oils, the future for using these compound as food preservatives lies in the careful selection and evaluation of their efficacy at low concentrations but in combination with other chemical preservatives or preservation processes. For this reason they are worth of study alone or in combination with processing methods in order to establish if they could extend the shelf-life of foods. In this study, the effect of the spices, clove, turmeric, cardamom, oregano, rosemary and garlic in controlling the spoilage and pathogenic bacteria is investigated. Their effect on biogenic amine formation in tuna especially, histamine, as a result of bacterial control is also studied in detail. The contribution of spice oleoresin in the sensory and textural parameters is investigated using textural profile analysis and sensory panel. Finally, the potential of spices in quality stabilization and in increasing the shelf–life of tuna during frozen storage is analysed

Pèptids biarílics a partir de 4-iodofenilalanina o 3-iodotirosina per borilació i reacció de Suzuki-Miyaura en fase sòlida. Avaluació de l'activitat antimicrobiana

En aquesta tesi doctoral es va estudiar la preparació de pèptids biarílics en fase sòlida. En primer lloc, es varen borilar residus de fenilalanina o tirosina presents a la seqüència peptídica a través d’una reacció de Miyaura. A continuació, es varen arilar els boronats resultants a través d’una reacció de Suzuki-Miyaura sota irradiació de microones, utilitzant diversos halurs d’aril i haloaminoàcids. La metodologia trobada es va estendre a la preparació de pèptids biarílics cíclics. Aquesta aproximació presenta l’avantatge d’evitar la síntesi en dissolució i la purificació del boronoaminoàcid. A més, permet la preparació d’una àmplia diversitat de pèptids biarílics a partir d’un únic boronopèptid. L’avaluació de l’activitat biològica dels pèptids sintetitzats va permetre idenficar seqüències actives enfront dels bacteris Erwinia amylovora, Xanthomonas vesicatoria, i Pseudomonas syringae, que són responsables de malalties greus en plantes d’interès econòmic com pereres i pomeres, i que varen resultar ser molt poc tòxics enfront cèl•lules eucariotes.

Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

Modulation of the human gut microflora towards improved health using prebiotics: assessment of efficacy

There is increasing awareness that the human gut microflora plays a critical role in maintaining host health, both within the gastrointestinal tract and, through the absorption of metabolites, systemically. An 'optimal' gut microflora establishes an efficient barrier to the invasion and colonisation of the gut by pathogenic bacteria, produces a range of metabolic substrates which in turn are utilized by the host (e.g. vitamins and short chain fatty acids) and stimulates the immune system in a non-inflammatory manner. Although little is known about the individual species of bacteria responsible for these beneficial activities, it is generally accepted that the bifidobacteria and lactobacilli constitute important components of the beneficial gut microflora. A number of diet-based microflora management tools have been developed and refined over recent decades including probiotic, prebiotic and synbiotic approaches. Each aims to stimulate numbers and/or activities of the bifidobacteria and lactobacilli within the gut microflora. The aim of this article is to examine how prebiotics are being applied to the improvement of human health and to review the scientific evidence supporting their use.

Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications

The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific populatiori groups and specific disease states. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Prebiotics

In nutritional sciences there is much interest in dietary modulation of the human gut. The gastrointestinal tract, particularly the colon, is very heavily populated with bacteria. Most bacteria are benign; however, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders. Bifidobacteria and lactobacilli are thought to be beneficial and are common targets for dietary intervention. Prebiotic is a non-viable food ingredient selectively metabolized by beneficial intestinal bacteria. Dietary modulation of the gut microflora by prebiotics is designed to improve health by stimulating numbers and/or activities of the bifidobacteria and lactobacilli. Having an 'optimal' gut microflora can increase resistance to pathogenic bacteria, lower blood ammonia, increase stimulation of the immune response and reduce the risk of cancer. This chapter examines how prebiotics are being applied to the improvement of human health and reviews the scientific evidence behind their use.

PASSCLAIM - gut health and immunity

Background The gut and immune system form a complex integrated structure that has evolved to provide effective digestion and defence against ingested toxins and pathogenic bacteria. However, great variation exists in what is considered normal healthy gut and immune function. Thus, whilst it is possible to measure many aspects of digestion and immunity, it is more difficult to interpret the benefits to individuals of variation within what is considered to be a normal range. Nevertheless, it is important to set standards for optimal function for use both by the consumer, industry and those concerned with the public health. The digestive tract is most frequently the object of functional and health claims and a large market already exists for gut-functional foods worldwide. Aim To define normal function of the gut and immune system and describe available methods of measuring it. Results We have defined normal bowel habit and transit time, identified their role as risk factors for disease and how they may be measured. Similarly, we have tried to define what is a healthy gut flora in terms of the dominant genera and their metabolism and listed the many, varied and novel methods for determining these parameters. It has proved less easy to provide boundaries for what constitutes optimal or improved gastric emptying, gut motility, nutrient and water absorption and the function of organs such as the liver, gallbladder and pancreas. The many tests of these functions are described. We have discussed gastrointestinal well being. Sensations arising from the gut can be both pleasant and unpleasant. However, the characteristics of well being are ill defined and merge imperceptibly from acceptable to unacceptable, a state that is subjective. Nevertheless, we feel this is an important area for future work and method development. The immune system is even more difficult to make quantitative judgements about. When it is defective, then clinical problems ensure, but this is an uncommon state. The innate and adaptive immune systems work synergistically together and comprise many cellular and humoral factors. The adaptive system is extremely sophisticated and between the two arms of immunity there is great redundancy, which provides robust defences. New aspects of immune function are discovered regularly. It is not clear whether immune function can be "improved". Measuring aspects of immune function is possible but there is no one test that will define either the status or functional capacity of the immune system. Human studies are often limited by the ability to sample only blood or secretions such as saliva but it should be remembered that only 2% of lymphocytes circulate at any given time, which limits interpretation of data. We recommend assessing the functional capacity of the immune system by: measuring specific cell functions ex vivo, measuring in vivo responses to challenge, e. g. change in antibody in blood or response to antigens, determining the incidence and severity of infection in target populations during naturally occurring episodes or in response to attenuated pathogens.

The potential of probiotics as in-feed growth enhancers for swine

Probiotics have enjoyed a surge of popularity in recent years, with many novel applications being proposed. One of the foremost for the agricultural industry is their potential for livestock growth promotion, a subject of special interest since the 2006 EU-wide ban on sub therapeutic levels of in-feed antibiotic growth enhancers. Probiotics work through a number of differing mechanisms, most of which are not, as yet, fully understood. The probiotics interact with the host’s natural gut flora in a complex and varying array of mechanisms, but ultimately work to improve nutrient digestibility and gut health and to suppress the actions of pathogenic bacteria. In conclusion, probiotics can be a useful replacement for in-feed antibiotic growth enhancers. However, care should be taken due to the variability of the size of the effect and the inconsistency of the results in the published literature.

Inhibition of Salmonella Typhimurium by tannins in vitro

Given the recent EU ban of antibiotics to promote the growth of farm animals, alternative approaches are needed for animal production systems. Tannins, which are already commercially marketed for animal nutrition, have bacteriostatic and bactericidal properties against pathogenic bacteria. The aim of this study was to investigate the inhibitory effect of various tannins against Salmonella Typhimurium (SL1344nal(r)) to identify potentially effective feed additives. Different sources of condensed and hydrolysable tannins were tested at concentrations between I and 6 mg ml(-1). The tannins tested were either commercial preparations or isolated from such preparations or from plants using Sephadex LH-20 based column chromatography. Some, but not all, of the tannins significantly decreased bacterial growth compared to tannin-free selenite cystine broth following incubation for 24 h at 37 degrees C. Gallotannins were especially effective and tara achieved 1.28 log(10) reductions after 24 hours. Antibacterial activity was also confirmed with inhibition zone diameters in a disc diffusion test. The experiments demonstrated that tannins may have potential as feed additives for reducing Salmonella infections in farm animals.

Proteomic analysis of triclosan resistance in Salmonella enterica serovar Typhimurium

Objectives: The aim of this study was to determine and compare the proteomes of three triclosan-resistant mutants of Salmonella enterica serovar Typhimurium in order to identify proteins involved in triclosan resistance. Methods: The proteomes of three distinct but isogenic triclosan-resistant mutants were determined using two-dimensional liquid chromatography mass separation. Bioinformatics was then used to identify and quantify tryptic peptides in order to determine protein expression. Results: Proteomic analysis of the triclosan-resistant mutants identified a common set of proteins involved in production of pyruvate or fatty acid with differential expression in all mutants, but also demonstrated specific patterns of expression associated with each phenotype. Conclusions: These data show that triclosan resistance can occur via distinct pathways in Salmonella, and demonstrate a novel triclosan resistance network that is likely to have relevance to other pathogenic bacteria subject to triclosan exposure and may provide new targets for development of antimicrobial agents.