National Drinking Water Clearinghouse
West Virginia University
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A Lesson in Microbiology
By Chain-Wen Wang, Ed.D., NDWC Contributing Writer and Craig Mains, Training Specialist, National Environmental Training Center for Small Communities
Editor’s Note: Many people will recall the 1993 cryptosporidium outbreak in Milwaukee, Wisconsin. An estimated one-fourth of the population there became ill and 100 people died as a result of drinking contaminated water. As dramatic as this case was, it may be only the tip of the iceberg. A 1987 U.S. Environmental Protection Agency report estimates that as many as 25 waterborne illnesses may occur for each one that is reported. The following article provides an overview of microbiology so that we may better understand waterborne illnesses and how to prevent them.
What are the types of microorganisms that can contaminate drinking water and cause illnesses and affect taste and odor? How do they differ from each other? To answer these questions, a very basic explanation of how biologists classify living organisms is necessary—with the understanding that classification schemes are constantly evolving.
A Micro-World Order
Until the mid-1800s, all living things were considered to be either plants or animals. Much of the general public still has this view. However, as scientists became more knowledgeable about the structure of microorganisms, it became clear that the two-kingdom classification was overly simplistic.
In 1866, Ernst Haeckel proposed a third kingdom, Protista, which included all microorganisms, such as bacteria, protozoans, and certain fungi and algae. Over time, though, scientists acknowledged that this three-kingdom classification was also inadequate.
In 1969, H.R. Whittaker of Cornell University proposed a five-kingdom classification of living organisms. Since then, due to many factors, such as electron microscopy and gene sequencing, scientists have proposed alternative seven- and eight-kingdom classifications. For now though, the five-kingdom system is commonly used and will be used here to discuss where different microorganisms fit in the scheme of classifications.
Bacteria—A fundamental biological distinction is made between bacteria and all other organisms. Bacteria (singular: bacterium) fall within the kingdom Procaryotae, also called Monera. A primary feature of bacterial or procaryotic cells is that their genetic material (DNA and RNA) is not contained in a membrane-enclosed nucleus.
Photo Source: www.photos.com
All other organisms, including fungi, protozoa, higher plants, and animals, are classified as Eucaryotes, which make up the other four kingdoms. Eucaryotes are generally larger than bacteria and are structurally more complex. All Eucaryotes have a membrane-enclosed nucleus, plus a number of other differences in cell structure.
While we often think of bacteria as agents of human infections, as a group they are probably the key to life on earth. One type of bacterium, the cyanobacteria or blue-green algae, has been found in fossils 3.5 billion years old and is considered to be responsible for creating an atmosphere in which humans and other higher plants and animals can live. Bacteria are the mediators of the carbon and nitrogen cycles and create conditions in soil that allow for plant growth.
Although they are often found in multi-cellular colonies or films, bacteria are single-celled, with each cell carrying out all of the cell functions. Typical bacterial cell sizes range from 0.20 to 2.0 micrometers. Bacteria have three basic shapes: (1) spherical, termed cocci (singular: coccus); (2) rod shaped, termed bacilli (singular:bacillus); and (3) spiral shaped. Bacteria can group themselves in distinctive ways. For example, cocci that form grape-like clusters are termed staphylococci and those that form chains are called streptococci
Potential waterborne bacterial pathogens include Vibrio cholerae, Campylobacter jejuni, Salmonella species, and Escherichia coli (E. coli). All of these organisms can secrete toxins that cause inflammation of the stomach and intestinal linings. E. coli gets a lot of attention because it belongs to the coliform group of bacteria that are universally used in standard tests as indicators of fecal contamination. E. coli strains vary widely in their pathogenicity. People tend to have a degree of immunity to local strains brought about by exposure. Visitors to an area lack that immunity, which can bring about cases of traveler’s diarrhea. Some strains of E. coli, however, such as E. coli 0157:H7, can cause hemorrhagic colitis that results in thousands of life-threatening illnesses and hundreds of deaths a year in the U.S., although most are associated with food contamination, not contaminated drinking water.
Photo Caption: This color-enhanced scanning electron micrograph shows Salmonella typhimurium (red) invading cultured human cells. Photo Source: Photo by Rocky Mountain Laboratories, NIAID, NIH with information from The National Institute of Allergy and Infectious Diseases
Protozoans—Protozoans belong to the kingdom Protista. Protista is a diverse grouping of organisms that includes certain algae, yeasts, slime molds, as well as the protozoans. Protista are one-celled organisms that are eucaryotic rather than procaryotic like bacteria. That is, they have their genetic material enclosed in a distinct membrane-covered nucleus. Protozoans have a flexible outer covering, rather than a semi-rigid cell wall like bacteria. The group includes organisms such as amoebas and paramecia.
Protozoans play key roles in the environment from participating in the decay of organic matter to constituting a large portion of plankton, which are free-floating organisms that are important links in aquatic food webs. Protozoans become dessicated easily and require damp or aquatic environments. They are generally much larger than bacteria, but there is some overlap between the largest bacteria and the smallest protozoans.
An important feature of some protozoans is that they are capable of producing cysts under adverse conditions, such as lack of moisture, food, or oxygen, or in the presence of toxic chemicals. In the case of parasitic protozoans, the cyst allows the organism to survive outside a host, which is crucial for those protozoans whose life cycle requires multiple hosts. Cysts present some challenges for water treatment because they are resistant to disinfection.
The protozoan Giardia lamblia (G. lamblia) is possibly the most common cause of waterborne diarrheal disease in the U.S. G. lamblia is commonly endemic in day-care centers, and more than five percent of adults and children are carriers who shed cysts in their feces, yet may have no symptoms. Infection is usually through cyst-contaminated water. Once cysts are ingested they transform themselves to an active feeding state called a trophozoite that attaches itself to the small intestine.
Photo Caption: This is a G. lamblia cyst in a stool.
Photo Source: www-medlib.med.utah.edu/parasitology/glambim.html
Other protozoans of concern include Cryptosporidium, Cyclospora, and Microsporidia. Cryptosporidium, which is widespread in the natural environment, is probably of greater concern than Giardia because its smaller size makes it harder to filter, and it is more resistant to disinfection.
Cyclospora and Microsporidia are recently emerging protozoal pathogens. The cysts of Cyclospora, which are bigger than those of Cryptosporidium, must be mature before they are infectious. So, if cysts are shed from the body before maturation, the host can avoid infections. Less is known about Microsporidia, and it is likely that cases may be underreported. Symptoms of protozoal infections include diarrhea, cramps, vomiting, and nausea. The extent of illness may be temporary for healthy people but can be fatal for immuno-compromised individuals.
Controlling Microbial Contaminants in Drinking Water
Because no water treatment technique is totally effective against all microbes—for example some microbes that may be resistant to disinfection may be effectively filtered—a multiple-barrier technique is commonly promoted. In fact, this strategy is required for systems using surface water or groundwater under the direct influence of surface water.
Multiple-barrier requirements include filtration and disinfection. Filtration treatment techniques can include rapid sand filtration, slow sand filtration, diatomaceous earth filtration, or various membrane filtration processes. Disinfection can be by conventional chlorination methods or by ultraviolet light or ozone processes. However, neither ultraviolet light nor ozone provides any residual disinfection capacity to control microbes in the distribution system.
In addition to filtration and disinfection, an effective multiple-barrier strategy should include good source water. This involves protecting and monitoring the watershed or aquifer that is the drinking water source. While a combination of filtration and disinfection is intended to reduce microbial comtaminants by 99 to 99.99 percent, if the water is of poor microbial quality, this level of removal may not be sufficient to protect public health. If certain protozoans or viruses are found in high enough concentrations in the source water, they may potentially breach the barriers of filtration and disinfection. Systems that use source water that is considered to be pristine may be able to waive the filtration requirements.
Maintaining sufficient pressure in the distribution system is also important in order to prevent the backflow of potentially contaminated groundwater into the lines. Finally, regular monitoring of coliforms and turbidity is necessary to ensure water quality and treatment effectiveness.
The U.S. Environmental Protection Agency has set drinking water standards for the following microbial contaminants: Cryptosporidium, Giardia lamblia, Legionella, enteric viruses, and total coliforms (which include fecal coliforms and E. coli). For each, there is a maximum contaminant level goal (MCLG) of zero. MCLGs are non-enforceable health goals. Except for total coliforms, the microbes mentioned are not required to be measured directly. Instead, a treatment technique, such as filtration and/or disinfection, is prescribed that is intended to remove or inactivate the contaminant by a set percentage.
Photo Caption: E. coli (red) adheres itself to intestinal cells.
Photo Source: Microscopy Consulting Services Inc, www.microscopyconsulting.com
Cryptosporidium is to be removed/inactivated by 99 percent, G. lamblia by 99.9 percent, and viruses by 99.99 percent. No percentage is set for Legionella, a bacterium that can lead to a type of pneumonia if it becomes aerosolized and inhaled, such as in a shower. The assumption is that if the other contaminants are removed or inactivated sufficiently, Legionella will also be controlled.
Total coliforms are required to be monitored directly with no more than five percent of samples testing positive in one month. Smaller systems that collect fewer than 40 samples per month can have no more than one positive sample. Any sample that tests positive for total coliforms must be re-analyzed for fecal coliforms and E. coli.
Heterotrophic plate count (HPC) is a rough measure of overall bacterial quality of water and is also regulated. HPC is a count of any bacteria that will grow on a general, non-selective medium. Water that has a high HPC count is bacterially suspect, and it is possible that high overall bacterial levels may interfere with total coliform tests, masking the detection of possible fecal contamination. There is no MCLG for HPC, and it is regulated by using treatment technologies that are expected to reduce the HPC to no more than 500 colony-forming units per milliliter.
Turbidity is a measure of the cloudiness |of water. Turbidity is a good measure of treatment effectiveness, because processes such as flocculation and filtration remove particles that contribute to turbidity. Because microbes may adhere to particles, water with high turbidity is microbially suspect. Microbes may be located in the interior of a clump of particles and be protected from disinfection. Turbidity is regulated by treatment technologies rather than direct measurement. However, instrumental measurement of turbidity at different stages of the treatment process and of the finished water is simple and economical.
Fungi and Algae—Fungi and algae are two diverse groups of organisms that present some difficulties in classifying. Fungi (singular: fungus) have eucaryotic cells, no chlorphyll, and obtain nutrition by absorbing soluble substances across their cell walls. Fungi that are multi-cellular fall into the Fungi kindgom, which includes mushrooms and most molds. Yeasts are one-celled fungi and are placed in the kingdom Protista. Some organisms that have traditionally been considered fungi, such as slime molds and water molds, have, with the advent of gene sequencing, been determined to be more closely related to other Protista than to fungi.
Photo Source: The Protist Server, mtlab.biol.tsukuba.ac.jp/WWW/Protist_menuE.html
Although water molds commonly grow on dead algae in freshwater environments, it is unclear to what extent fungi affect water treatment systems. Some are known to grow on walls of distribution lines and treatment tanks and have been detected in finished water. However, their main impact is generally considered to be on taste and odor, and there is little information available on their health effects.
The term “algae” has no officially recognized biological meaning. It originally referred to any simple aquatic plant. Most biologists now use algae (singular: alga) to refer to organisms that have no true roots, stems, or leaves but have chlorophyll and are capable of photosynthesis. Depending on whether an alga is unicellular or multi-cellular, it is considered either part of the Protista kingdom or Plant kingdom, respectively. There is also no apparent consensus on whether algae, by definition, must be eucaryotic. If procaryotes with chlorophyll that are capable of photosynthesis, such as the cyanobacteria (blue-green algae), are included, then algae are found scattered throughout three kingdoms.
Largely viewed mainly as the cause of taste, odor, and color problems, algae, especially the cyanobacteria Microcystis, Cylindrospermopsis, and Anabaena, have recently come under more scrutiny in the U.S. for their health effects. Cyanobacteria produce toxins that, depending on the filter media in use and the concentration of the toxin in the source water, can result in undesirable concentrations in finished water. The cyanotoxins, microcystin, and cylindrospermopsin are linked to nerve and liver damage. Blooms of blue-green algae are generally seasonal and are limited to surface waters.
Algae and their toxins are currently unregulated by the Safe Drinking Water Act. However, they are included on EPA’s Drinking Water Candidate Contaminant List (CCL), which is a list of known drinking water contaminants that may require future regulations. Although something of an emerging issue in the U.S., cyanotoxins have been recognized as a global health issue for some time. Australia has a regulatory program in place and the World Health Organization has a 1.0 microgram per liter guideline for microcystin.
Viruses—Viruses fall into an entirely different category, being totally unlike any of the organisms just mentioned. In fact, they may not qualify to be called organisms. All living organisms contain both DNA and RNA. Viruses may have DNA or RNA, but not both and, unlike true living organisms, their nucleic acids are inactive outside of host cells. On the other hand, once they enter host cells, their nucleic acids become active, and they are able to replicate. They also are able to cause infection and diseases just like bacteria, fungi, and protozoans.
Photo Caption: This is a slide of a type B viral Hepatitis as shown in its chronic stage.
Photo Source: Transplant Pathology at the University of Pittsburgh, tpis.upmc.edu/
Viruses in general are much smaller than bacteria and can be viewed only by electron microcroscopes. However, there is some overlap in size between the largest viruses and the smallest bacteria. Their small size and resistance to environmental stress creates water treatment challenges. Viruses are less easily filtered by soil than bacteria, and viruses have been detected in groundwater that was absent of fecal indicator bacteria.
Enteric viruses are viruses that infect the gastrointestinal tracts of humans and animals. There are more than 140 known types of human enteric viruses, many of which cannot be cultured. Infected individuals can excrete large numbers of viruses in their feces (one billion viruses per gram of feces), even though they may be asymptomatic.
Enteric viruses that have been associated with waterborne disease outbreaks include Hepatitis A virus, Hepatitis E virus, Norwalk and Norwalk-type viruses, Rotaviruses, and Enteroviruses. Enteroviruses are a large group that includes Polioviruses and Coxsackieviruses.
Typical symptoms of viral infections include diarrhea, nausea, vomiting, abdominal pains and cramps, and fever. Symptoms are usually self-limiting in healthy individuals, but compromised individuals are at risk for life-threatening illnesses. Some enteric viruses are linked to respiratory symptoms, liver infections, central nervous systems infections, and chronic fatigue syndrome.
Because it is difficult to test for the presence and concentration of viruses, water systems in the U.S. that use surface water or groundwater that is directly under the influence of surface water are required to use a combination of treatment options and disinfection that will result in a four-log (99.99 percent) reduction or inactivation of viruses. Recognizing that a four-log reduction in viruses might not provide reliable protection from infection for systems that use poor quality source water, EPA has promulgated additional requirements, including sanitary surveys, although not all are required of small systems.
Chain-Wen Wang and Craig Mains are married to each other and are the proud parents of Corbin Alexander, born in 2003. They are active with watershed groups, including the Downstream Alliance, in northern West Virginia.
The following Web sites provide additional information about microbiology:
• www.germology.com has basic facts about microbes, written for the layperson;
• EPA’s Office of Ground Water and Drinking Water site has information about the Surface Water Treatment Rule and the Total Coliform Rule at www.epa.gov/OGWDW/mdbp/mdbp.html;
• The Center for Disease Control has information on pathogenic microbes under the Health Topics A-Z menu item at www.cdc.gov;
• More about cyanobacteria may be found on the World Health Organization Web site at www.who.int/docstore/water_sanitation_ health/toxicyanobact/begin.htm.