Analysis of the fine specificity and cross-reactivity of monoclonal anti-lipid A antibodies

We have investigated the fine specificity of anti-lipid A antibodies to identify conserved lipid A antigens. Because lipid A derived from many different Gram-negative bacteria has similar biologic activities, the conserved regions may be of particular importance for the immunostimulatory and toxic properties of lipid A. We found that five of nine antibodies bound to a wide variety of Gram-negative bacteria. All these widely cross-reactive antibodies bound to the same antigenic site within lipid A. Polymyxin B, an inhibitor of lipid A activity, bound to this site as well. The widely cross-reactive antibodies bound to native and base-hydrolyzed lipid A equally well, and also bound to the monosaccharide precursor lipid X. The less cross-reactive antibodies recognized base-hydrolyzed lipid A poorly, and did not recognize lipid X at all. Other investigators have shown that lipid X has some of the activities of lipid A in vitro and can inhibit the lethal toxicity of LPS in vivo. On the basis of this study, we suggest that lipid X contains a conserved lipid A epitope as well.     

Metabolic activity of bacterial cells enumerated by direct viable count.

The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included [methyl-3H]thymidine or [U-14C]glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.     

Frequency and characteristics of plasmids in bacteria isolated from deep-sea amphipods

Bacterial strains isolated from deep-sea amphipods were identified, classified, and screened for plasmid content. Plasmids were common, with 11 of 16 isolates carrying one or more plasmids; these ranged in size from 2.9 to 63 megadaltons. Several of the strains demonstrated distinctly different phenotypic traits yet contained plasmids of the same molecular weight. Results of agarose gel electrophoresis, DNA hybridization, and restriction analysis indicate that the plasmids detected in these deep-sea isolates are identical, suggesting that transmission may occur in the deep-sea environment and that plasmids are common in some deep-sea habitats.     

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The current study evaluated the effect of FMRFamide on both light and serotonin induced phase shifts of this rhythm. The application of FMRFamide was found to block serotonin induced phase shifts but, by itself, FMRFamide did not cause significant phase shifts. Furthermore, the effects of FMRFamide on light-induced phase shifts appeared to be phase dependent (i.e., the application of FMRFamide inhibited light-induced phase delays but actually enhanced the magnitude of phase advances). As in Aplysia, the eye of Bulla gouldiana also contains a circadian pacemaker. In Bulla, FMRFamide prevented light-induced phase advances and delays. Although FMRFamide alone generated phase dependent phase shifts, it did not cause phase shifts at the phases where it blocked the effects of light. These data demonstrate that FMRFamide can have pronounced modulatory effects on phase shifting inputs to the ocular pacemakers of both Aplysia and Bulla.Abbreviations ASW artificial seawater - CAP compound action potential - CT circadian time - 5-HT serotonin     

Biodiversity amongst microorganisms and its relevance

Concluding remarks from the joint IUBS/IUMS workshop on Biodiversity amongst microorganisms and its relevance held in Amsterdam on 7–8 September 1991. An international microbial ecology programme can be justified in its own right now that appropriate investigative tools have been developed. Microorganisms influence global change, and indicate global health and environmental quality. At the same time, an inventory of the world's microbial species and their properties is required, together with associated culture collections and genomes. Sampling methods need to be standardized, both for species and functions. Extreme environments are a particularly rich source of microbial genomes, and endangered habitats should be sampled as a matter of priority. Cataloguing and conserving the world's microbial biodiversity is justifiable and scientifically important.     

Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples.

Sewage effluent and outfall confluence samples were collected at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico; outfall confluence samples at Ocean City, Md., were also collected. Samples from uncontaminated open ocean areas served as clean-water controls. Bacteria were enriched in marine broth 2216 amended with 1 microgram of one of a set of chemicals selected for study per ml: nitrobenzene, dibutyl phthalate, m-cresol, o-cresol, 4-nitroaniline, bis(tributyltin) oxide, and quinone. MICs of the chemicals were determined individually for all isolates. Bacterial isolates were evaluated for resistance to nine different antibiotics and for the presence of plasmid DNA. Treated sewage was found to contain large numbers of bacteria simultaneously possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin, erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples, but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic sewage-impacted waters or from uncontaminated open ocean sites.     

Effects of microcosm salinity and organic substrate concentration on production of Vibrio cholerae enterotoxin.

The effects of aquatic processes on production of cholera toxin by Vibrio cholerae were studied with seawater microcosms. Several salinity and organic nutrient concentrations were employed. At 10 g of organic nutrient per liter of seawater, toxin production increased as the salinity was increased. At lower organic nutrient concentrations, toxin production was markedly enhanced when the salinity was 20 and 25%. Toxin concentration increased with salinity, independent of cell concentration and toxin stability. From the results obtained in this study, it is concluded that physical and chemical parameters of the aquatic environment affect not only the physiological state of V. cholerae, but also its potential pathogenicity.