Research Project 2: Microbial Pathogens in Tropical Coastal Waters: An Ecosystem Approach to Determine Risk and Prevent Water-Borne Diseases

Roger S. Fujioka, Ph.D., Project Leader
Philip C. S. Loh, Ph.D., Co-Investigator
Alan R. Katz, M.D., Co-Investigator
Steven E. Seifried, Ph.D., Co-Investigator
Grieg Steward, Ph.D., Co-Investigator
Alan D. Tice, M.D., Co-Investigator
Bruce A. Wilcox, Ph.D., Co-Investigator
Vijay Kannapan, Ph.D., Research Associate
Audrey Asahina, Research Assistant
Amy Henry, Research Assistant
Olivia Nigro, Research Assistant
Dayna Sato, Research Assistant
Gayatri Vithanage, Research Assistant

See Publications at bottom of this page.

Identification of Microbial Water Quality Problems in Tropical Climates

The policy of EPA is to establish environmental standards and apply these standards equally to every state without regard to differences in environment of the various states. Hawaii is the only state with a tropical climate and two lines of evidence show that microbial populations behave differently in tropical environments. As a result, EPA water quality standards developed from data obtained from temperate climate are not reliable in tropical climate. The first line of evidence is that all streams in Hawaii contain concentrations of EPA-approved fecal indicator bacteria (fecal coliform, E. coli, enterococci) that exceed EPA recreational water quality standards. EPA water quality guidelines assume that fecal matter is the only significant source of these fecal indicator bacteria and when water quality standards are exceeded, that body of water is significantly contaminated with sewage and the risk for transmission of sewage borne pathogens is unacceptable. However, the tropical soil environment in Hawaii and other tropical locations (Guam, Puerto Rico, south Florida), support the growth of EPA-approved fecal indicator bacteria and they become established in soil as a significant environmental and non-fecal source of these bacteria. Rainfall washes these soil-bound fecal bacteria into streams at high concentrations, which no longer indicate degree of fecal contamination. Thus, one identified problem is the need to develop appropriate water quality standards for tropical climates. The second line of evidence that tropical climates can affect microbial populations in environmental waters is greater prevalence of three non-enteric pathogens (Leptospira sp, Staphylococcus aureus, Vibrio spp) in tropical waters, which cause higher incidences of water borne diseases in Hawaii than in the rest of the states. Significantly, water borne transmissions of these three pathogens are not recognized as high risk in temperate climate of continental USA and EPA has not established water quality guidelines or methods for these pathogens. As a result, the second identified problem is the need to develop methods to establish water quality guidelines for water borne transmission of these three non-enteric pathogens that occur more frequently in tropical climates.

In summary, the two identified water quality problems in Hawaii (appropriate fecal microbial water quality standards, increased incidence of some non-enteric water borne diseases) are directly attributable to changes in microbial populations in tropical climates and EPA policies, which directs all states to adopt the same water quality management strategy without regard to environmental differences in different climates.

Approaches to Solve the Water Quality Problems in Tropical Climates.

Accept the fact that tropical climates cause significant changes in the environment of coastal waters as compared to temperate climates and therefore data obtained from tropical climates will be required to solve tropical microbial water quality problems.

As a working hypothesis, accept the principle of microbial ecology, which states that microbial populations and microbial processes in environmental waters are controlled by environmental conditions and when there is a significant change in the environment (tropical versus temperate climate), the sources, survival and fates of the microbial populations will change.

Assess all the major factors that affect coastal water quality and determine which factors control the prevalence of water borne pathogens and indicators of pathogens. In this regard, traditional factors such as how people use environmental waters for recreation and the differential impacts of point sources (sewage) and non-point sources of discharges that contaminate various coastal water sites will be determined. For this study, samples will be obtained from selected coastal water sites of the islands of Hawaii. The potential sampling sites will include: a) All sites used by the state Department of Health to monitor the quality of recreational waters as these are sites where humans use and are exposed to coastal waters. b) All of the monitoring sites used by government and private agencies to assess the impact of ocean sewage discharge to recreational waters. c) All coastal water sites designated by the Department of Health where water quality has been compromised due to discharge of streams and storm drains. d) Stream and coastal water sites which have been previously characterized (eg. UH, USGS) and where some water quality data already exists e) Reclassification of streams draining from a hydrological watershed based primarily on land use, anthropogenic activities and ecological measurements of organisms in streams and their predicted impact on receiving coastal water sites.

A team of seven researchers with diverse and complementary expertise will work together to design appropriate experiments to address the major objective of this study, which is to obtain data and information to assess reliable relationships between water quality measurements, pathogens in tropical coastal waters, and risk assessments for water borne disease transmission. This will be accomplished by obtaining data utilizing EPA approved culture methods, using innovative methods and using newer molecular and gene probe methods to obtain necessary data, which cannot be obtained by the EPA approved methods. Recognizing that recovery of pathogens from coastal waters may not be sufficient to conclude that human infections are transmitted by coastal waters, a comparison (physiological, immunological, genetic) will be made between the pathogens recovered from coastal waters with those recovered from human infections along with incidence of infections by these pathogens among human populations.

Apply molecular methods and ecological approaches to address the theoretical problem that in warm tropical coastal water ecosystems, exchange of virulence genes between bacterial populations may occur with greater frequency.

Address EPA’s need for data documenting the presence and prevalence of human enteric viruses in coastal waters to support their conclusion that human enteric viruses are responsible for most of the diseases related to recreational use of waters. To address this need, this study will use filter-feeding mollusks (Isognomon), which are indigenous to coastal waters of Hawaii to bioconcentrate viruses from coastal waters.

Goals and Objectives of Proposed Study

The immediate goal of this study is to determine the effects of tropical climates on the sources, prevalence and public health significance of pathogens and indicators of pathogens in tropical coastal waters. The long-term goal of this study is to develop water quality guidelines and standards, which are reliable for tropical coastal waters and which will improve the health of people living in the tropics. Recognizing the complexity in reaching the goals of this study, four sub-projects have been developed. The objectives and the leaders of these four subprojects are listed below:

Sub-Project 1

Establishing appropriate water quality guidelines for tropical waters.
Principal Investigator: Roger Fujioka, Ph. D. (Water Quality and Public Health Microbiologist).

1. Determine the sources, survival and ecology of enteric pathogens (Salmonella, adenoviruses) and non-enteric pathogens (Staphylococcus aureus, Vibrio spp), as well as traditional fecal indicators of pathogens in water and sediment at selected coastal water sites.
2. Determine which alternative indicator of water quality (Clostridium perfringens, FRNA coliphages, Bifidobacter, Bacteroides by PCR, enterococci by culture and PCR, fecal sterols, Vibrio spp., S. aureus) can be reliably used to predict contamination by enteric and non-enteric pathogens.
3. Characterize the relationships between indicators of pathogens and pathogens recovered from coastal waters with those recovered from clinical samples for the purpose of establishing reliable water quality guidelines for tropical waters.

Sub-Project 2

Application of an innovative method of using indigenous mollusk (Isognomon) to bioconcentrate human enteric viruses and other pathogens from selected coastal water sites.
Principal Investigator: Philip Loh, Ph.D (Animal and Aquaculture Virologist).

1. To optimize the method of using filter-feeding mollusks indigenous to Hawaii (Isognomon) to bioconcentrate pathogens, especially human enteric viruses, from tropical waters and in this way overcome the limitations of using mechanical devices to concentrate and recover viruses from coastal waters.
2. To use molecular methods to supplement the limitations of culture methods to determine for the presence of all relevant human enteric viruses.
3. To characterize the pathogens (viruses, bacteria) recovered from coastal waters and to compare with reports of clinical isolates.

Sub-Project 3

Assessment of tropical coastal marine ecosystems as reservoirs of virulence genes for bacterial pathogens (Staphylococcus aureus, Vibrio spp).
Principal Investigator: Grieg Steward, Ph.D. (Microbial Ecologist).

1. To determine the presence and abundance of virulence genes within DNA extracted from bacterial and bacteriophage assemblages in seawater, sediments and biofillms at selected coastal sites around Oahu.
2. To characterize the virulence gene complement of specific pathogens (S. aureus, Vibrio spp) cultivated from coastal waters for comparison with those isolated from clinical samples.
3. To isolate and characterize bacteriophages infectious for S. aureus and pathogenic Vibrio spp. in coastal water ecosystems in order to evaluate their possible use as control agents.

Sub-Project 4

Integration of ecological and public health approaches to determine and predict health risks associated with recovering pathogens from coastal waters.
Principal Investigaor: Alan Tice, MD, (Infectious Diseases in Human Populations), Alan Katz, MD. (Medical Epidemiologist), Bruce Wilcox, Ph.D (Watershed Ecologist and Human Health Effects ), and Steven Seifried, Ph.D. (Specialist in Bioinformatics, Biochemical and Molecular Methods).

1. Use of public health approaches to assess risk for transmission of diseases by pathogens isolated from coastal waters based on comparing virulence properties of pathogens recovered from human infections and from coastal waters.
2. Characterize watersheds based on ecology and anthropogenic activities and their predicted impact on quality of receiving coastal waters.
3. Application of bioinformatics and molecular methods for data storage, data management and characterization of pathogens.

Publications

C. D. Sinigalliano, M. L. Gidley, T. Shibata, D. Whitman, T. H. Dixon, E. Laws, A. Hou, D. Bachoon ||, L. Brand, L. Amaral-Zettler, R. J. Gast, G. F. Steward, O. D. Nigro, R. Fujioka, W. Q. Betancourt, G. Vithanage, J. Mathews, L. E. Fleming, and H. M. Solo-Gabriele. 2007. Impacts of Hurricanes Katrina and Rita on the microbial landscape of the New Orleans area. Proc. Natl. Acad. Sci. USA , 10.1073/pnas.0610552104.
http://www.pnas.org/cgi/content/abstract/0610552104v1

Identification of Prophages in Coastal Water Isolates of Staphylococcus aureus.  Michael Workman, Olivia D. Nigro, and Grieg F. Steward.  In Press.

Virulence and Antibiotic Resistance Gene Combinations among Staphylococcus aureus Isolates from Coastal Waters of Oahu, Hawaii.  Stephanie N. Stotts, Olivia D. Nigro, Tonya L. Fowler, Roger S. Fujioka, and Grieg F. Steward.  July 2005.