Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment

Conditions influencing the survival of Campylobacter jejuni in the natural aquatic environment have been determined. Release of Campylobacter spp. into natural waters by animal hosts is postulated to play a key role in the maintenance of viability and transmission of the organism in the environment. Laboratory flask microcosms containing filter-sterilized stream water were used to test C. jejuni for the ability to remain viable in simulated natural systems. The microcosms were compared with the biphasic and shaking broth procedures used routinely for growth of Campylobacter spp. in the research laboratory. The stream-water microcosms were analyzed to determine effects of temperature and aeration on the survival of a well-characterized C. jejuni strain isolated originally from a human campylobacteriosis patient. Morphological characteristics were evaluated by phase-contrast microscopy and scanning or transmission electron microscopy. Survival curves were quantified on the basis of plate counts, epifluorescent microscopy, optical density measurements, and direct viable counts associated with protein synthesis in the absence of DNA replication. A significant difference was observed between results of direct enumeration, i.e., direct viable counts or acridine orange direct counts, and those from spread plate cultures. In all cases, increasing temperature of cultivation resulted in decreased recoverability on laboratory media, due possibly to an increased metabolic rate, as analyzed by CO2 evolution in the presence of radiolabeled glutamate. Stream water held at low temperature (4 degrees C) sustained significant numbers of campylobacters for greater than 4 months. Microcosms, aerated with shaking, exhibited logarithmic decline in recoverable C. jejuni, while stationary systems underwent a more moderate rate of decrease to the nonculturable state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial-invertebrate interactions and potential for biotechnology

Conclusion As the interactions between marine invertebrates and their bacterial commensals and symbionts are better understood, the application of biotechnology will enhance both environmental and economic benefit. In the immediate future, marine bacteria, either selected or genetically engineered, will play a significant role in enhancing the development of selected invertebrates in aquaculture and in the field. Luck may also favor discovery of mechanisms to suppress the development of biofouling species, perhaps by making it possible to coat submerged surfaces with bacterial films designed to repell larvae and/or interfere with larval morphogenesis. In any case, the future is appealing.     

Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

The potential of biotechnology for developed and developing countries

Summary The extraordinary developments occurring in biotechnology throughout the world will lead to significant changes in world commerce, as well as in human health and welfare. A few dramatic advances have already been made in medical and agricultural biotechnology. Production of vaccines employing recombinant DNA methods is one example of the potential for genetic engineering applications in the treatment of hereditable and communicable diseases. Lesser understood, but with equally dramatic promise, is marine biotechnology, an example where the excellent possibilities for industry applications in both developing and developed countries can be elucidated. Recent successes in the cloning of growth hormone genes in salmon and other important genes and gene complexes in fin fish and shellfish promise major changes in production of commercially important fish and shellfish. Pharmacologically active compounds from marine animals and plants can now be harvested effectively, using biotechnology. Specialty chemicals and enzymes with unusual properties, unique to the marine environment, with potential for industrial applications, are being discovered. Thus, new products and new markets appear on the horizon. What has changed the picture significantly is the ready access which is now available to these new materials and compounds by the application of the methods of genetic engineering. No longer is it necessary to depend on sustained harvest, which may be subject to vagaries of weather or climate. By means of genetic engineering, genes coding for economically desirable traits can be cloned and those systems established in the laboratory in controlled culture and production. The enormous natural resources of developing countries cannot be productively harnessed by the developing countries themselves, since the industrial resources needed for the technology and the trained intellectual potential are not in abundant supply. Partnerships between developed and developing countries should be established so that the benefits of biotechnology can be achieved by both. An important role for applied microbiology, thus, is described and a dramatic impact at the global level can be predicted if effective biotechnology partnerships between the developing and developed countries of the world are created.

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Resumen El extraordinario desarrollo de la biotecnología en todo el mundo va a generar importantes cambios tanto en el comercio mundial como en la salud y bienestar de la humanidad. En las áreas de biotecnología médica y agrícola se han realizado importantes progresos. Un ejemplo de las posibilidades de la ingeniería genética en el tratamiento de enfermedades hereditarias y contagiosas es la produccion de vacunas utilizando técnicas de recombinación de ADN. La biotecnología marina es un área menos conocida pero no por ello con menos posibilidades de aplicaciones industriales tanto en países en desarrollo como en los desarrollados. Los éxitos obtenidos recientemente en la clonación de genes que codifican para las hormonas de crecimiento del salmón, así como de otros genes y conjuntos de genes de distintos peces y moluscos, suponen cambios importantes en la producción comercial de pescados y mariscos. Compuestos farmacológicamente activos procedentes de plantas y animales marinos pueden ahora cosechar se más eficientemente gracias a la biotecnología. Se estan descubiendo compuestos químicos y enzimas con propiedades especiales, unicas en el ambiente marino, cuya aplicación industrial tiene un futuro prometedor. Así pues, nuevos productos y nuevos mercados aparecen en el horizonte. La facilidad de acceso ahora disponible a estos nuevos materiales y compuestos, mediante la aplicación de los métodos de la ingeniería genética, ha cambiado significativamente el panorama. Ya no se ha de depender de cosechas sujetas a las variaciones del tiempo y del clima. Mediante la ingeniería genética los genes que codifican para características económicamente deseables pueden clonarse y los sistemas así obtenidos establecerse en laboratorio en condiciones de cultivo y producción controladas. La cantidad de recursos naturales que poseen los países en desarrollo no pueden ser utilizados productivamente por ellos mismos debido a la escasez de desarrollo tecnológico y potencial intelectual de los mismos. Deberián establecerse colaboraciones entre países en desarrollo y países desarrollados de manera que tanto unos como otros puedan beneficiarse de los progresos en biotecnología. Es este un papel importante que puede tener la microbiología aplicada y se puede predicir, a nivel global, un enorme impacto si se crean colaboraciones eficientes entre países en desarrollo y países desarrollados.

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Résumé Les extraordinaires développements de la biotechnologie dans le monde modifieront de façon significative le commerce mondial, aussi bien que la santé et le bien-être de l'homme. Des progrès considérables ont déjà été accomplis dans le domaine de la technologie médicale et agricole. La production de vaccins par les méthodes de l'ADN recombinant est un exemple du potentiel du génie génétique pour le traitement des maladies héréditaires et transmissibles. On connait moins le potentiel tout aussi considérable de la biotechnologie marine où on trouve d'excellents exemples d'applications industrielles réalisables aussi bien dans le pays développés que dans les pays en voie de développement. Les succès récents, obtenus par clonage des gènes d'hormones de croissance chez le saumon et par clonage d'autres gènes et groupes de gènes chez les poissons et les crustacés, laissent prévoir des changements majeurs dans le domaine de la production des poissons et crustacés d'intérêt commercial. Grâce à la biotechnologie, des composés pharmacologiquement actifs peuvent être obtenus à partir d'animaux et de végétaux marins. Des produits chimiques et des enzymes doués de propriétés nouvelles et spécifiques au milieu marin ont été découverts et sont suscéptibles d'applications industrielles. Ainsi, de nouveaux produits et de nouveaux marchés pointent à l'horizon. Ce qui a changé le tableau de façon décisive, c'est la facilité avec laquelle ces nouvelles substances peuvent être obtenues par les méthodes du génie génétique. Il n'est plus nécessaire d'avoir recours à des récoltes exposées aux aléas climatiques. Les gènes codant pour des caractères économiquement désirables peuvent être clonés au laboratoire et utilisés pour une production en cultures contrôlées. Faute de disposer des moyens technologiques et du potentiel scientifique nécessaires, les pays en voie de développement ne peuvent pas utiliser par eux-mêmes leurs énormes ressources naturelles. Des coopérations entre pays développés et moins développés doivent s'établir de façon à ce que la biotechnologie soit mutuellement profitable. La biotechnologie aura ainsi un rôle important et dont on peut attendre des effets considérables à l'échelle mondiale, à condition toutefois d'instituer une coopération effective entre les pays hautement industrialisés et le Tiers Monde.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16.8.1985. Opening Session     

Ecological relationships between Vibrio cholerae and planktonic crustacean copepods.

Strains of Vibrio cholerae, both O1 and non-O1 serovars, were found to attach to the surfaces of live copepods maintained in natural water samples collected from the Chesapeake Bay and Bangladesh environs. The specificity of attachment of V. cholerae to live copepods was confirmed by scanning electron microscopy, which revealed that the oral region and egg sac were the most heavily colonized areas of the copepods. In addition, survival of V. cholerae in water was extended in the presence of live copepods. Attachment of viable V. cholerae cells to copepods killed by exposure to -60 degrees C was not observed. Furthermore, survival of V. cholerae was not as long in the presence of dead copepods as in the live copepod system. A strain of Vibrio parahaemolyticus was also seen to attach to copepod surfaces without effect on survival of the organism in water. The attachment of vibrios to copepods was concluded to be significant since strains of other bacteria, including Pseudomonas sp. and Escherichia coli, did not adhere to live or dead copepods. Attachment of V. cholerae to live copepods is suggested to be an important factor of the ecology of this species in the aquatic environment, as well as in the epidemiology of cholera, for which V. cholerae serovar O1 is the causative agent.     

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats

Abstract— Seven-day-old rats were injected intraperitoneally with l -phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain. The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain. Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell-free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.     

Microbial degradation of model petroleum at low temperatures

Two areas of Chesapeake Bay, Colgate Creek in Baltimore Harbor and Eastern Bay, are presently under study, with routine sampling of water and sediment for petroleum-degrading microorganisms (bacteria, yeasts, and fungi) by direct plating and enrichment culture. Selected physical and chemical parameters are recorded for each sampling site, and water and sediment samples are extracted for hydrocarbons. Numbers of petroleum-degrading microorganisms enumerated by direct plating were found to correlate with the concentration of benzene-extractable material and were higher for the Colgate Creek than for the Eastern Bay site. Petroleum-degrading microorganisms were isolated from water and sediment samples at environmental temperatures of 0°, 5°, and 10°C. A salts medium supplemented with nitrate and phosphate was used to provide optimum conditions for petroleum degradation, whereas Chesapeake Bay water was used to simulate natural environmental conditions. Use of a model petroleum permitted quantitative measurement of utilization of individual hydrocarbons ranging in complexity from simple alkanes to polynuclear aromatic hydrocarbons. Higher growth yields and maximum hydrocarbon degradation was observed for microorganisms in the salts medium at 0°, 5°, and 10°C, although significant quantities of hydrocarbons were utilized in some samples grown in a medium for which Chesapeake Bay water was the diluent. Bacterial hydrocarbon degradation accounted for most of the model petroleum utilization at 0° and 5°C. However, oscillations of bacterial populations, with significant growth of yeasts, was observed at 10°C. Photomicroscopy and scanning electron microscopy revealed aggregates of bacteria, yeasts, and fungi associated with oil globules. From preliminary identification and classification of the hydrocarbon-utilizing bacteria, members of the generaVibrio, Aeromonas, Pseudomonas, andAcinetobacter were present in the enrichment cultures. From results of this study, it is concluded that utilization of model petroleum at low temperatures is a function of the types and numbers of microorganisms present in an original inoculum taken from the natural environment.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.