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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Microbial Contamination Jed Fuhrman I first would like to echo the critical importance of knowing the basic properties of the marine system, as Dr. Morel discussed. Although most of my topic here is contamination—that is, things that we have been adding to the marine environments and problems with those additions —it is going to be absolutely essential that we understand how that marine system works before we could understand how we could stop or solve some of the contamination problems. I briefly discuss that subject, but mostly I cover the subject of microbial contamination of marine environments. I attempt to define it and briefly describe what we are doing now and what we can do in the future to improve it. When I use the term contamination, I mean a release of microorganisms into the environment, usually from released waste products. People use incredible amounts of water. Most of it goes through pumped systems. It becomes mixed with human waste and all kinds of other waste and is dumped back into the water cycle and out into the ocean. A primary concern that most people have is human safety related to disease; but of course, many of us are also very concerned about degrading the habitats in natural systems. Microbes that cause contamination include bacteria (relatively small [~1 mm linear dimension] cellular prokaryotes) and viruses that are non-cellular and very small (~30 to 200 nm). The viruses do not metabolize without their hosts, which is very important because it denotes that they McCulloch-Crosby Chair of Marine Biology, University of Southern California, Los Angeles, CA
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP are very different from bacteria, which reproduce and do things on their own. In addition to bacteria and viruses, protists can sometimes be disease organisms, as Dr. Burkholder will discuss. We do not know very much about degradation of the habitats of natural systems from microbes that we are releasing out there, compared with release of, for example, nutrients or chemicals. There might be some very serious problems in that lack of knowledge. Mostly what we know about is when we put a microbe in the environment and it comes back to us as a possible disease agent, and that is the primary subject of my talk. EXPOSURE TO CONTAMINANTS Our main potential exposure is from eating shellfish such as clams and mussels. In filtering huge amounts of water (much better than most filters we make), they filter many microbes that we release into the environment, which are subsequently funneled back to us through these organisms, if eaten. I am not going to talk much about shellfish testing, but it is a very serious concern to many people, mostly handled by federal and state food safety agencies. We are exposed to microbial contaminants when we have contact with the water, such as during swimming, surfing, and boating. A few people have also talked about aerosols, sometimes seen as a haze on the windshield of a parked a car at the beach. It is the oily residue from the sea surface that comes out as sea spray, and there are actually many aerosols that come out of the marine environment from surf and spray. There is some possible concern about aerosols causing microbial contamination, but very little work is being done on that subject. IMPACT OF CONTAMINANT EXPOSURE The financial impact of even the perception of microbial contamination in the marine environment is extremely costly because people spend a lot of money to go to the beach, live near the beach, or be involved with recreation somehow associated with the beach. The affected industries include tourism, real estate, and a multitude of support industries, which might be far from the beach itself, such as a place in the Midwest that makes boogie boards, beach towels, or floats. The total economic input that relates to use of the beach is in the billions of dollars. SOURCES OF CONTAMINATION The one source of contamination that immediately comes to mind, sewage treatment plant effluent, is actually quite well regulated. People
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP know in general what is coming out from sewage treatment plants, and on the West Coast it tends to be released from some deep pipe offshore (excuse my West Coast slant). In the marine environment on the East Coast, there are typically estuaries, and much of the treated sewage may proceed through an estuary out to sea. Thus, although there is a great deal of close-to-shore exposure, relatively few people go swimming in many of the major estuaries near where the sewage comes out (like New York Harbor). Although sewage from treatment plants is probably not the most serious exposure problem (but still a concern, especially regarding shellfish), we are actually more concerned these days about runoff and nonpoint sources—rivers or storm drains, the latter especially on the West Coast. These sources are known to be a real problem. A related concern is coastal septic systems, where there is no local sewer system and people's individual septic systems have a connection through ground water to the sea. From these sources, contaminated water is released into the marine environment directly at the shoreline or at an estuary —precisely where people want to go swimming. It is rarely regulated, very poorly understood, and comprises a very large amount of material. I am aware of only one epidemiology study in which people swimming at the beach in Los Angeles were examined (Haile and others 1999). It provides evidence that swimming in a storm drain compared with 400 m away results in approximately twice the likelihood of getting certain symptoms. Many of the symptoms, which include intestinal ailments, rashes, respiratory problems, and fevers, probably come from viruses and not just bacteria (relevant because of the kind of testing that is done today). TESTING AND DETECTION OF CONTAMINATION Water Quality Testing agencies measure certain kinds of viable bacteria by growing them on Petri dishes: They usually pour 100 mL of water through a filter, put it on a dish with growth medium, and see what grows in 24 hours. Therefore, results require a full day. They usually count bacteria called total coliforms, fecal coliforms, and/or enterococci. Standards are a threshold of allowable bacteria of the various types, and sometimes a ratio is used as well. Such a ratio was recently adopted in California. If the count or ratio exceeds a threshold, then the authorities take some sort of action such as posting the beach (posting warnings) or actually closing the beach to swimming. Different places have different responses to detected contamination.
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP I will mention the California threshold values and put these numbers in context in a subsequent discussion of how many native marine bacteria are already in the water. The new California thresholds are (1) total coliforms at 105 colony forming units (CFU) per liter, (2) fecal coliforms at 4 × 104 CFU per liter, (3) enterococci at 104 CFU per liter, or (4) a ratio of total fecal coliforms less than 10 when total coliforms exceed 104. Interestingly, these standards were adopted in large part because of the epidemiology study finding that the inclusion of the ratios covered a higher incidence of illness. This set of standards is one of the few with a real scientific basis. Many older standards were more arbitrary, although they were developed to be as scientific as possible. For example, if an “average” person can become ill from swallowing 1000 bacteria, and will swallow × amount of water swimming, the standard should be such and such. These calculations were difficult or impossible to verify. The epidemiology study verified some of these calculations. These bacteria are considered indicators because most of the organisms that grow on these plates are not pathogens themselves. Although one would not want to eat the microbes growing on the plates, one could be exposed to many of them and not become ill. They are simply indicators of microorganisms that probably came from feces, to which one would not want to be exposed. The problem with indicators in the context of bacterial testing is that we know many of these coliforms may be coming from animal sources such as birds. So if you have an estuary that happens to be in a bird corridor, with birds migrating through in large numbers, test results will indicate significant amounts of bacteria in the water. Sometimes those bacteria are not human pathogens and not indicators of human problems; they are an indicator that there have been birds or, in some cases, sea lions or something like that. Although it makes sense to avoid water with significant amounts of animal feces, it is not clear what sorts of illnesses are being avoided or if the same standards should apply. So these indicators are not perfect by any means. Viruses I previously mentioned the testing of bacteria at the beach; however, viruses are not routinely monitored at beaches in the United States. I think there are a few places in Europe that are just starting to do it. A classical, “standard” way that people measure viruses is to sample a large quantity of water (~100 gallons) and put it through a charged filter. In seawater the salts shield the charges, so it is necessary to coagulate or otherwise treat the viruses to be caught by the filter. The viruses are
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP extracted into a small volume, the extract is added to a tissue culture of something like monkey kidney cells, and after 1 or 2 weeks (depending on the particular viruses), one looks for plaques where the kidney cells have been killed. Each typically represents one original virus (or a small clump of them). This process takes some time, but it detects viable pathogenic viruses. These are viruses that will kill those kidney cells; like the bacteria test, this method finds the viable ones. However, there is no established standard for viruses in recreational water. No one knows how many per 100 gallons is safe. More importantly, the long time to obtain results does not help in making a decision to close the beach. If it takes 2 weeks to do a test, it is not helpful to say, “Two weeks ago we should have closed the beach.” For this reason, the standard method is not a very practical management tool. However, a very important issue is that when we talk about detection of virus, we have the proverbial “needle in a haystack.” We talked about 105 per liter of certain kinds of pathogenic bacteria. One might talk about very small numbers of viruses: A few per liter could be harmful because one ingested virus can cause an illness. With bacteria, it usually takes hundreds or thousands to cause an illness. Thus, when we talk about this, we say that there is a lot of natural background here; and when we talk about detection, we have the needle-in-a-haystack situation. Typically bacteria are 109 per liter and viruses are 1010per liter. A photomicrograph taken by epifluorescence microscopy of stained viruses and bacteria (see Fuhrman 1999) graphically demonstrates the abundance, which looks like stars on a very clear night. The big dots are bacteria, and all of the little dots everywhere are viruses. Note that this is from non-contaminated seawater, 10 miles offshore in deep water. These are just the naturally occurring viruses and bacteria. We do not know what kind of viruses these are, although they seem very important in natural ecological and biogeochemical processes (Fuhrman 1999). We are just starting to identify the kinds of bacteria using new molecular techniques to which Dr. Morel alluded, and we are finding that some of these are not even bacteria. In surface waters, 5% of them are archaea, and in the deep sea, maybe 50% are archaea; and they group phylogenetically with the thermophilic species. The closest cultured relatives to the marine archaea have an optimum temperature of 105°C, and they like hot acid. But these archaea are living in seawater—highly aerobic conditions, cool, normal salt, and so forth. They are not like any other archaea that anyone knows about, which I think is fascinating and important. My main interest is to study these bacteria and these viruses because I believe it is not possible to study the contaminants by themselves in the marine environment without studying the surrounding native organisms. The processes that bacteria use in nature to defend themselves against
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP viruses are probably what is killing off the viruses that we send out to the environment. Recent Advances in Detection There have been few changes in the tests for bacteria such as Escherichia coli, one kind of fecal coliform. Most of the current tests still require growth for 24 hours, which is still remarkably simple and inexpensive. In California, the sampling is often done by lifeguards; it is not something that requires a huge amount of training, and it is not very expensive or difficult to do. You simply need to have small filter units that are sterile, show someone how to carry out sterile technique, allow the bacteria to grow in incubators, and count which organism is of a certain color on a plate. There have been significant advances in virus tests. It is now possible to look for their genetic material without waiting for them to grow. The reverse transcriptase polymerase chain reaction (RT PCR) can be used to look for specific pathogens and a variety of viruses in marine environments. The following viruses have been found in marine environments: (1) enteroviruses, which are polio, coxsackie, and echoviruses; (2) hepatitis A; (3) adenovirus; (4) Norwalk virus; and (5) rotavirus. These viruses cause a veritable laundry list of illnesses. The tests are reasonably fast (about a day) but costly (about $1000 per assay if you add up all the costs), and they require highly trained operators. I estimate that there are one dozen people in the United States who could probably perform the test without additional training or experience. Many more could learn, if given a detailed protocol. There are only a few published reports of using this method with marine samples, one being that of Griffin and colleagues (1999), who found a great deal of contamination in the Florida Keys. One unresolved question is whether the tests measure nonviable viruses. In other words, because the test is looking at their genetic material, it is not known to what extent there is dead RNA lying around. There probably is some, but RNA is rather labile stuff. It does not tend to persist very long, but it could survive. So perhaps to some extent, it is an indicator. Why then do we not simply rely on these bacterial indicators because, after all, these are just another problem resulting from fecal contamination. Should they not all go together in the water? You release this stuff in the water. Perhaps all the problem components follow each other around like tracers; that is the way an engineer might first think about it. However, unlike nonbiological tracers that move with the water, viruses and bacteria each have their own physical and biological properties, and they are quite different. Bacteria can repair damage from sunlight. Even though E. coli might not thrive in seawater and will probably even-
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP tually starve there, if you damage a cell, its repair mechanism could repair it even in seawater. Bacteria might divide also in a marine environment, depending on the conditions; they could be eaten by protists. Viruses cannot repair their damage because they do not have metabolism. They could adsorb or desorb from particles in ways very different from the bacteria. They could remain pathogenic for months under cold, dark conditions. People have found them in the Northeast, for example, in cold sediments; pathogenic viruses from sewage could last for 1 or more years in a viable condition. In our laboratory, we have been using RT PCR with samples from Southern California beaches to detect enteroviruses. We have approximately 50 measurements from which we have compared detection of viruses with the standard bacterial test. We find there is very little relationship between them—neither a correlation nor any statistically significant relationship. They do not appear to follow each other. Our results (Noble and Fuhrman 2000) suggest that under some cases, it would be prudent to test for both bacteria and viruses and not just bacterial indicators, as is done today. Virus testing might be considered first at high-use beaches adjacent to storm drains, for example. However, we are not yet ready for that because several aspects require more work; rapid concentration methods of viruses from seawater are necessary. Virus detection methods must be less expensive and more rapid, but still specific and simple. They probably should be made quantitative, which we are starting to do now. If possible, they should also have an indicator of viability. Consider the current test. Instead of simply taking seawater and running the RT PCR test directly, we start with 20 L of seawater, filter it through a 0.2-µm filter, and then put that filtrate through a concentration unit that filters all the water away but leaves the viruses behind. This process holds particles greater than about 30 nm. The procedure currently takes several hours and ends up with about 50 µl of all the virus-size material from that 20 L of seawater. We run the RT PCR test on a few microliters of that material, followed by gel electrophoresis (about 1 hour). In the end, comparison with positive and negative controls allows us to interpret positive or negative results. Sometimes we find there is some other material in the concentrate that interferes with some of the tests, making them inconclusive (negative with the natural sample, but still negative when the authentic virus is added to the concentrate). Future Improvements This whole procedure might be improved by the development of rapid methods to concentrate large volumes of water and maintain high
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP sensitivity. Detection may be sped up with some possible approaches such as probes, called molecular beacons. These probes are internal to the PCR product and have a quencher. When they are bound to their target, they unquench, becoming fluorescent when the target is present. Such probes can be used in the PCR test and detected while the test is being done, allowing for a quick quantitative answer. Taqman is another similar test, and it relies on enzymatic release of a fluorophore from a labeled oligonucleotide probe. Looking farther into the future, an instant test would be a huge help. Most of you probably have seen that a physician can rub a throat swab on a small plate and test for strep throat in a few seconds. It used to take a day, with sample transport to a remote lab and labor-intensive tests. Imagine if a similar test allows lifeguards to roll an instrument around on the beach, testing for bacteria and maybe even for viruses at different locations. They might sample a few liters, put it in a machine, and get quantitative answers in minutes. It might be possible to develop tests like that for seawater, with sufficient resources invested. The same machine could test drinking water, reservoirs, rivers, among other possibilities, and could also be used to track sources of contamination. It is not hard to imagine it being cost-effective, but the initial investment in development is the difficult part. Another major need regards standards: We need to know what level is safe and what levels are unsafe; when to close the beach and when not. Our knowledge is greatly lacking in such situations. An obvious possible move in this direction would be more epidemiology studies coupled with measurements of these viruses and bacteria. Most importantly, we need to understand more about the factors that control microbial contamination. Even if we could be certain about closing a beach, it is more important to be able to learn what is happening and how to mitigate the problems. How do we change the way we are releasing pathogens into the environment—the timing, location, or something—so that we might be able to solve some of these problems? How do we know when it might be safe to go back to the water after it has been closed without having to run these tests, especially if they continue to be expensive? To answer all of these questions, we need to know much more about what controls these pathogens once they are released into the environment. Regulations now use what might be called engineering-like approaches. Some treat microbes as a conserved component like salinity. In fact, there are new standards, using so-called total maximum daily load (TMDL), that seem to be a great improvement regarding what can be released into the environment. However, in Santa Monica Bay in Los Angeles, for example, they have a single number for the coliforms in the
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP whole bay. The standard calls for a certain amount of coliforms, or less than a certain level, as if the bay is one place; yet you can walk along the beach and get a 10-fold change in coliforms over 100 m in some cases. Obviously, some improvements are still needed. We must be able to look at variability and patchiness, because regulation of these microorganisms requires understanding more about how they are moving and what kind of processes are controlling them. ADDITIONAL ISSUES One additional issue regards questions relating to pathogens of marine organisms. Although this is not my main area of expertise, I have heard people talk about epidemics among marine organisms, possibly including endangered species such as marine mammals. Almost certainly these are exacerbated by pollution or some other source that might have stressed the immune system of these animals. As a separate issue, there is the question of marine organisms as a reservoir for human or terrestrial animal diseases. Here we are talking about viruses or perhaps bacteria that have a terrestrial animal source, which then enters the marine environment, infecting marine organisms and then returning to infect land organisms, possibly including humans. Usually, viruses have one species of host or closely related hosts. Some viruses, however, jump from host to host, such as from pigs to humans. Such jumping to or from marine animals is very poorly understood. Some recognized broad host-range examples include the caliciviruses that include Norwalk-like viruses. Some are reported to have remarkably wide host ranges, even including fish and mammals for certain serotypes. More work is needed before we can say whether the exact same strain jumps that far and might infect humans. Recent examples of viruses jumping to marine mammals include reports of canine distemper in seals in Europe. If epidemics of marine mammals become more severe, one might imagine a higher probability of infections that jump across species lines. It might become a zoonotic concern to the human population if there is a reservoir in a marine environment that keeps reinfecting something that affects humans. As our population increases and moves closer to the coast, this virus jumping could worsen any problem that might exist. REFERENCES Fuhrman JA. 1999 Marine viruses: Biogeochemical and ecological effects. Nature 399:541-548.
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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Griffin DW, Gibson CJ, Lipp EK, Riley K, Paul JH, Rose JB. 1999 Detection of viral pathogens by reverse transcriptase PCR and of microbial indicators by standard methods in the canals of the Florida Keys. Appl Environ Microbiol 65:4118-4125. Haile R, Witte J, Gold M, Cressey R, McGee C, Millikan R, Glasser A, Harawa N, Ervin C, Harmon P, Harper J, Dermand J, Alamillo J, Barret K, Nides M, Wang G. 1999 The health effects of swimming in ocean water contaminated by storm drain runoff. Epidemiology 10:355-363. Noble RT, Fuhrman JA. 2000 Enteroviruses detected by reverse transcription polymerase chain reaction in the coastal waters of Santa Monica Bay, California: Low correlation to bacterial indicator levels. Hydrobiologia (Forthcoming).
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