By Kelly A. Reynolds, Ph.D.
Recently, you’ve most likely heard about the human genome project in the media— science’s international effort focused on mapping the entire human genome.1 Knowledge of organizing human genetic building blocks promises to provide needed information on the basic structure and function of the body while enabling researchers to target areas of disease origin, diagnostics, treatment and therapy. Did you know the same technology is also being applied to waterborne pathogens? Researchers have now determined the complete genomic sequence of the agent responsible for cholera—a potentially fatal waterborne disease striking in epidemic proportions worldwide.
Decoding pathogen secrets
Genome sequencing has increased in popularity in recent years with the advent of new molecular methodologies that enable scientists to determine the basic genetic make-up of just about any organism—from bacteria to humans. Once the genetic building blocks are mapped, one can begin to evaluate possible evolution of the organism and its relation to other species—location of genes that code for fatal or debilitating diseases (i.e., cancer genes) or traits that enable increased survival and persistence (i.e., antibiotic and chlorine resistance).
In the case of many bacteria, a variety of strains exist. Some, or even the majority, aren’t harmful. A good example is the bacterium, E. coli. For decades, all strains of this organism were considered harmless indicators of fecal contamination, indicative of the possible presence of other pathogens also found in feces. Interest in this organism has peaked since several virulent strains have been associated with both food and water outbreaks (i.e., strain O157:H7) resulting in numerous fatalities, primarily in children and the elderly—most notably in Walkerton, Ontario, in May and a county fair in upstate New York last summer.
In light of the emergence of new pathogens and increased bacterial resistance to antibiotics, genetic sequencing tools provide new information on the life of a pathogenic organism that may aid in development of new antibiotics and vaccines. Sequence data also provides new ways of looking at the evolutionary history of bacteria. Unfortunately, whole genome sequencing is a daunting task. Led by Dr. Frederick R. Blattner of the University of Wisconsin in Madison, the genetic mapping of one strain of E. coli took nearly six years and the collective efforts of more than 250 scientists.2
Genetic analysis of the pathogenic region of the E. coli showed related sequences to Shigella, a known pathogen. Portions of the pathogenic Shigella organism may have integrated with the harmless E. coli to create a new pathogen. We know that organisms in the environment can “share” genetic information and this sharing of genes may enable a new pathogen to emerge or become particularly resistant to treatment. Understanding, through molecular characterization studies, how these genes are transferred—and at what frequency—will enable researchers to better develop strategies for preventing the emergence or transmission of these pathogens. Only a handful of bacteria have been entirely sequenced, but the trend is increasing.
Sequencing V. cholerae
Recently, Vibrio cholerae has been added to the list of organisms for which the complete genomic sequence is available.3 The complete sequence was determined from a representative isolate of the ongoing seventh pandemic of cholera in Asia. The V. cholerae genome is composed of more than four million DNA base pairs, requiring the expertise of more than 30 researchers to fully sequence. The organism has a diverse natural habitat, can be attached to zooplankton, exists in the water column in a free-floating state and may also act as a human pathogen in the gastrointestinal tract. Genetic mapping of V. cholerae revealed areas coding for several pathogenic factors including toxin production and intestinal colonization. Evaluation of the sequence information may aid researchers in determining how a commonly found, free-living marine organism can also act as a human pathogen.
The cholera pandemic
Hundreds of thousands of people are sickened by V. cholerae throughout the world. In 1999 alone, 220,000 cases were reported resulting in more than 8,400 deaths. The current seventh pandemic began in 1961 in Indonesia. The disease rapidly spread to Asia and Bangladesh in 1963. By 1966, the epidemic reached India, the USSR, Iran and Iraq. Cases occurred in Africa in 1970, after an absence of over 100 years of the disease.
Peru was struck in 1991 and the disease has spread throughout South America. The epidemic in Latin America is proving to be difficult to contain due to the lack of modern infrastructure, water and wastewater treatment methods and a safe, clean supply of drinking water. In addition, since coastal waters are natural reservoirs for the organism, it’s unlikely that exposures to the pathogen itself will ever be eliminated. The number of countries currently affected approaches 120 and continues to increase. From 1997 to 1998, there was a surge of cholera cases, with the total number nearly doubling. The disease is rare in the United States (0-5 cases per year) or in countries where sanitation is good and water supplies are pure or treated.
Cholera is transmitted by contaminated food or water. The organism may be present in feces or vomit of infected individuals. Thus, contamination events may be direct from the natural environment or indirect from the feces of an infected individual. Even those unaware that they’re infected may continue to propagate spread of the disease since the organism may be found in feces for 7-to-14 days after infection. The disease tends to spread rapidly in areas with poor sanitation. The bacterium survives well in brackish and marine waters and outbreaks have been associated from raw or undercooked shellfish.
Cholera infections are often mild or produce no symptoms at all; however, approximately one in 20 persons suffers severe symptoms involving profuse watery diarrhea, vomiting and leg cramps. If treatment isn’t issued immediately, death can result within 24 hours, killing up to 50 percent of those infected. Treatment for cholera involves rehydration with oral rehydration solution or intravenously. Antibiotics may increase recovery time but isn’t necessary for successful treatment. Vaccines are currently available for V. cholerae but are only effective 50 percent of the time and, at best, last only six months.
V. cholerae can survive in a variety of foods for up to five days at room temperature and longer when refrigerated. Freezing prevents proliferation of the organism but doesn’t kill it. The organism is however sensitive to acid environments (i.e., carbonated beverages) and drying. Cholera infections may be avoided by drinking only treated water and ice (i.e., boiled, chlorine, iodine, reverse osmosis, etc.), and cooking foods thoroughly. Raw vegetables should be peeled and raw or undercooked seafood should be avoided as should foods and beverages from street vendors. The World Health Organization offers this simple rule: “boil it, cook it, peel it, or forget it.”
Like E. coli, V. cholerae includes both pathogenic and nonpathogenic strains that can be differentiated by examination of their genetic maps. Certain strains are also severe pathogens for fish and mammals. They’re indigenous inhabitants of marine environments, whose presence doesn’t necessarily indicate a contamination event. An important trait of the organism is that it’s able to enter a viable but nonculturable state under certain environmental conditions and thus becomes undetectable by conventional cultural methods, while still capable of initiating disease. Evaluation of genetic differences amongst strains may provide further clues as to the physiological changes and the emergence and survival of this puzzling organism.
Genetic sequencing’s future
The aim of genetic sequencing is to understand the evolution and basis of pathogenicity and environmental persistence of various organisms. In addition, how organisms exchange information and to what end they can emerge as new pathogens is of interest. Finally, new vaccines and medicines may be developed that can better address the prevention of epidemic infections of V. cholerae. Complete genome sequencing may at first initiate more questions than answers. Although the enormous task of sequencing the organism is done, it’s only a starting point for countless other research endeavors. An understanding of what the genomic traits infer shall remain topics of further investigation for decades to come.
Currently, the University of Wisconsin Genome Project lists approximately five more pathogenic E. coli strains that are being sequenced in their entirety in addition to a strain of Shigella flexneri (cause of dysentery), Salmonella typhi (cause of typhoid fever) and Yersenia pestis (cause of plague).4 As information unfolds regarding the implications of the human genome map, molecular technologies promise to continue to reveal new insights into waterborne bacteria and other pathogenic organisms and how they relate to human health.
- Human Genome Project Information, Oak Ridge National Laboratory, Oak Ridge, Tenn.: http://www.ornl.gov/hgmis/elsi/elsi.html
- Blattner, F.R., et al., “The complete genome sequence of Escherichia coli K-12,” Science, 277(5331)1453, 1997.
- Heidelberg, J.F., et al., “DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae,” Nature 406, 477-483, 2000.
- University of Wisconsin Genome Project home page: http://www.genomewisc.edu
About the author
Dr. Kelly A. Reynolds is a research scientist and microbiologist at the University of Arizona with a focus on the development of methods for detecting human pathogens in drinking water. She also has been a member of the WC&P Technical Review Committee since 1997.
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