This accomplishment-based renewal of the Pacific Research Center for Marine Biomedicine (PRCMB) will be a collaboration between the University of Hawaii, Stanford, and Jackson State University. Research activities will be focused on two areas: (1) pathogens in tropical marine waters and (2) marine toxins and pharmaceutical lead discovery. This new research follows logically from accomplishments and discoveries made during the first phase of PRCMB funding.
The pathogen and pathogen indicator work will focus on two streams that discharge to the ocean, one tropical (Kaelepulu on the island of Oahu) and the other temperate (San Pedro Creek in California). Monthly sampling at the two streams will be used to characterize seasonal variability in pathogen indicators and pathogen (bacterial and viral) concentrations using both culture-dependent and culture-independent (PCR-based) methods. These studies will be complemented by high-frequency sampling (ten-minute and hourly) during storm and dry-weather conditions to provide insights concerning spatial and temporal variability. Health-risk models (Quantitative Microbial Risk Assessment) will be developed using pathogen and pathogen indicator concentrations as input.
The marine toxin work will concern ciguatoxin and beta?{methylamino-L-alanine (BMAA), both of which were the focus of recent PRCMB studies. Taking advantage of (1) the refinement of an assay (n2a) that is now capable of detecting ciguatoxin in fish tissue at concentrations ten times below the threshold associated with ciguatera symptomology and (2) a collaboration with Hawaiian recreational fishermen, PRCMB scientists will extract and concentrate sufficient ciguatoxin from fish tissue to obtain a molecular structure and will then, in collaboration with scientists at the University of Washington, work toward the development of an antibody-based assay for ciguatoxin. Ciguatoxin research will also involve (1) exploratory refinement of the n2a assay based on generation of nitric oxide by the n2a cell line and (2) an investigation of environmental conditions that trigger ciguatoxin production by taxonomically defined strains of the dinoflagellate Gambierdiscus. The BMAA work will build on initial studies to determine the prevalence of BMAA in marine cyanobacteria and the transfer of this toxin to higher trophic levels. The pharmaceutical studies will take advantage of the more than 2,500 microbial isolates in the PRCMB culture collection that have not yet been screened for bioactivity. Extracts of these cultures will be used in cell-based and molecular assays to determine if they affect the growth of microbial pathogens (Candida albicans, Escherichia coli, Staphlyococcus aureus, multi-drug resistant S. aureus, and vancomycin resistant Enterococcus faecium) or if they exert effects on human adenocarcinma cells, on protein kinase C, or on mitogen-activated protein kinase. Identification of the compounds responsible for reproducible bioactivity in these extracts will be accomplished through bioassay-guided fractionation and spectroscopic analysis (e.g., mass spectrometry, high-field nuclear magnetic resonance spectrometry)
The broader impacts of the proposed work fall into several categories. One is certainly the public health endpoints: (i) credible methods that can be used to determine whether coastal waters are safe for recreational use, (ii) a simple, inexpensive assay for ciguatoxin in fish, (iii) a better appreciation of the threat to human health associated with the movement of BMAA through marine food chains, and (iv) the discovery of novel compounds with application to the treatment of human health problems. Second, this is a collaborative proposal with an historically black university (Jackson State University) and a female principal investigator from Stanford. Finally, the PRCMB will continue to train students and to collaborate with other institutions and Ocean and Human Health centers.
The study investigated sources and transport of pathogens in coastal waters and health affects associated with exposure to them. Oahu, Hawaii and California were chosen as study areas to investigate the effects of stream discharge on pathogens in coastal waters. On Oahu, we enumerated human viruses and bacterial pathogens, as well as common water quality indicators in 22 streams. We found viral and bacterial pathogens in a number of streams. We used a quantitative microbial risk assessment to then determine the risk of acquiring gastrointestinal illness from exposure to the stream water once it had discharged to the coastal ocean. We found that the model-predicted risk was correlated to concentrations of Clostridium perfringens, which is a bacterial water quality indicator. In California, we studied the fate of stream discharge in the coastal ocean at Pacifica State Beach. We developed a method of predicting how much of stream discharge would be entrained in a surf zone where people may come into contact with it. This is important because stream discharge can contain human pathogens. We discovered during our work that beach sands may also be an important source of pathogens and pathogen indicators to coastal waters. We characterized the microbial community in beach sands from 49 beaches along the California coast and determined which organisms including potential pathogens in the sand could be readily eluted and transported into the surf zone. We also investigated which bacteria in human sewage are most resistant to sunlight inactivation in seawater. This project involved significant outreach efforts to the USEPA, state-level department of health and water quality agencies, as well as the general public during field studies. The work involvedgraduate students and post doctoral scholars. During the field work at Pacifica, dozens of undergraduate students helped during the experiments.
