Waterborne Pathogens

Now seems like an ideal time to have an overview of viruses in water.

By Ed Butts, PE, CPI

Figure 1. Cell structure of a single-celled (prokaryotic) organism.

When I originally planned my columns for 2020, I had no idea the world would be in the grip of a crisis. As I write this in March, the World Health Organization (WHO) just proclaimed the coronavirus to be a worldwide pandemic; the Centers for Disease Control and Prevention (CDC) has endorsed the practice of social distancing; and travel is largely halted all around the world. Who knows what it will be like when you read this in June?

Although it does not appear that the transmission of the current affliction is necessarily or primarily related to the use or consumption of water, it occurred to me we can never take this subject lightly. For the good of our customers and ourselves, we must always be cognizant of the identification, risks, and prevention of waterborne pathogens—the subject of this month’s column.

In general, the greatest microbial risks are associated with the ingestion of water that is contaminated with human or animal feces. Wastewater discharges that occur in freshwaters and coastal tidal or seawaters are the major source of fecal microorganisms, including harmful pathogens.

Although they can occur anywhere, the risk of outbreaks of waterborne diseases increases where standards of water, sanitation, and personal hygiene are low. Thus, acute microbial diarrheal diseases are a major public health problem in developing and third-world countries as those affected by diarrheal diseases usually have the lowest financial resources and poorest sanitary facilities. Children under five, primarily in  Asian and African countries, are the most affected by microbial diseases transmitted through water.

However, microbial waterborne diseases also affect other countries. For example, in the United States it is estimated that 560,000 people suffer from severe waterborne diseases and 7.1 million suffer from mild to moderate infections, which results in an estimated 12,000 deaths a year.

The Basics

Although many species of microorganisms exist in all natural waters, they generally perform mostly beneficial processes.However, certain bacteria or other microorganisms, such as pathogenic protozoa or viruses, may cause illness in humans.

Within the basic groups of microorganisms, there are three subgroups in water chemistry: non-pathogenic, pathogenic, and opportunistic.

Obviously, many microorganisms have adapted to survive in, on, or within the human body over the past centuries. Many of these microorganisms cause no bodily harm nor result in illness—and are even beneficial because they directly compete with the other microorganisms that could cause disease if they become established or grow in or on human bodies. These microorganisms are included in the non-pathogenic group.

The next group, the pathogenic group of microorganisms include fewer microorganisms than the non-pathogenic group, but they can potentially cause disease in humans. Some of these pathogenic microorganisms are closely associated with humans and other warm-blooded animals and their waste products.

These pathogens are often transmitted from one living organism to another through direct or intimate contact or food or water contamination. Cells of the pathogen are shed from the host in the fecal matter, and if these same cells further propagate to contaminate food or water consumed by another person or animal, the disease spreads and the cycle continues.

A final group, opportunistic pathogens may not be closely associated with or normally cause illness in humans or other mammals. Therefore, they rarely cause disease in healthy adults. Instead, these may be commonly occurring bacteria or fungi which naturally exist in soil or water.

However, they can end up being disease-causing organisms in persons already weakened by a pre-existing disease or an immune-suppressed condition, such as AIDS, Lupus, or liver/kidney disease.

The Pathogens

Waterborne pathogens are microorganisms present in both groundwater or surface water supplies that can cause disease such as typhoid fever, salmonella, giardiasis, and other gastrointestinal illnesses.

Pathogens are diverse organisms and consist of both prokaryotic organisms, a single cell that lacks a membrane-bound nucleus (Figure 1), and eukaryotic organisms, whose cells have a nucleus enclosed within a membrane.

The four groups of pathogens in water of greatest concern include bacteria, viruses, protozoa, and parasitic worms. Among these, the most known and prevalent pathogens are bacteria and viruses. While both can cause infectious disease, bacteria and viruses are different in both their structure and function.

Bacteria

Bacteria exist in many shapes and sizes—from minute spheres, cylinders, and spiral threads to flagellated rods and filamentous chains.

Pathogenic bacteria are prokaryotic cells that cause disease by producing toxins and can occur in surface water or groundwater sources in small or large numbers. They do so by either occurring in excretion from feces or naturally from the local environment. Typically, they range in size between 0.5 and 2.0 micrometers (microns).

Bacteria are divided into three types based on their response to gaseous oxygen. Aerobic bacteria require oxygen for their health and existence and will die without it. Anaerobic bacteria cannot tolerate gaseous oxygen at all and will die when exposed to it. Facultative bacteria prefer oxygen but can live without it.

Figure 2. Cell structure of an animal virus.

Common disease-causing bacteria that can be transmitted by water supplies include Campylobacter jejuni, Vibrio cholerae, Escherichia coli, and Salmonella, among other pathogenic bacteria shown in Table 1a and Table 1b.

Viruses

Viruses vary widely in size and shape and are the smallest of all waterborne pathogens as they typically range in size from 0.03 to 0.10 microns. Viruses are not living organisms, and are therefore unable to replicate without a host cell.

Viruses are particles of nucleic acid (DNA or RNA) encased within an outer protein shell or capsid (coat). A virus has a tail which it attaches to the bacterium surface by means of proteinaceous pins or spikes (Figure 2). The tail contracts and the tail plug penetrates the cell wall and underlying membrane, injecting the viral nucleic acids into the host cell.

There are predominantly two kinds of shapes found among viruses: rods/filaments and spheres. The rod shape is due to the linear array of the nucleic acid and the protein subunits making up the capsid. The sphere shape is actually a 20-sided polygon.

Viruses cause disease by taking over their host’s cell machinery to reproduce numerous copies of the virus. This activity destroys the host cell in the process. There are more than 100 known types of human and animal enteric viruses that may be readily transmissible through natural water sources. Viral diseases with the potential for water transmission include Coronavirus, Hepatitis A, Hepatitis E, and Norovirus (Table 2).

Protozoa

The protozoan group are single-celled organisms known as amoebas or eukaryotes. Members of protozoans that are potential waterborne pathogens include Amoebiasis, Cryptosporidium parvum, Giardia lamblia, and Naeglaria fowleri. (Table 3).

There is a greater range in physical size among the protozoan group of pathogenic organisms, usually between 1-5 microns for Cryptosporidium up to 10 to 15 microns for Giardia. Therefore, filtration must accomplish removal down to 1-2 microns for Cryptosporidium and 3-5 microns for Giardia.

Currently, enteric protozoa cysts such as Giardia lamblia (Figure 3) and Cryptosporidium parvum (Figure 4) can lead to the gastrointestinal illnesses known respectively as giardiasis and cryptosporidiosis. These are considered as the most important and difficult protozoan pathogens in water quality, particularly since ingesting as few as 10 cysts can lead to illness.

Although these protozoa are most found in surface water sources, they can also be present in shallow groundwater sources and from wells with broken casings or inferior seals.

Many protozoan infections that are mild in normal individuals can be life-threatening in immune-suppressed patients, particularly patients with acquired immune deficiency syndrome (AIDS).

They each represent challenges to conventional water treatment as they form thick-walled outer shells that can survive for long periods of time in the environment, can readily pass through media filtration without coagulation, and are also resistant to common disinfectants, such as chlorine.

Parasitic Worms

Although not as common as bacteria or viruses, parasitic worms can also be present in a water source and cause disease and illness in humans. These include tapeworms, pinworms, helminths, schistosomiasis, and trichinosis. Adult worms live in the intestines and other organs.

Figure 3. Giardia lamblia cyst.

Many of the pathogens that are spread through potable water are also spread through other common means of transmission, including direct or airborne contact between people who are infected, contact with infected animals, contaminated food, and exposure from swimming in contaminated pools.

Both parasitic worms and enteric protozoa are eukaryotic pathogens and parasites. A parasite is a general term for any living organism that lives in or on another living organism. It may feed off its host or obtain shelter using its host, but contributes nothing to its host’s well-being.

Human parasites include fungi, protozoa, and worms. Many of the pathogens which cause gastrointestinal disease shown in Figure 5 are included in this category. Several human (i.e., host) gastrointestinal pathogens produce toxins which act on and in the small intestine, causing a secretion of fluid into the gut. This usually results in diarrhea, and in turn, often leads to severe dehydration.

Detecting Viruses

As the size of viruses are so small, they generally require microscopic examination for detection. The fecal indicator bacteria group (including Escherichia coli, fecal coliforms, and fecal streptococci) are typically used to indicate the sanitary quality of water for water supply purposes.

The fecal indicator bacteria group are natural and productive inhabitants within the gastrointestinal tracts of humans and other warm-blooded animals and normally cause no harm. They are released into the environment with fecal matter and are then exposed to a variety of environmental conditions that eventually cause their demise.

In general, it is thought that the fecal indicator group cannot thrive in natural environments since they were adapted over generations to survive in the warm gastrointestinal tract. Studies have shown that fecal indicator bacteria may survive from a few hours up to several days in surface water to many days or even months in lake sediments where they are protected from the ultraviolet rays provided from direct sunlight as well as most predators.

The stable temperature in groundwater as well as competition for nourishment with other non-pathogenic bacteria found naturally in the water, predator action by protozoa and other small organisms, natural aquifer flocculation and filtration processes from slow velocity, and physical entrapment in minute pore spaces—all these contribute to their demise.

As it is assumed that other pathogens similar to the fecal indicator bacteria die at the same basic rate as this group of bacteria, if relatively high numbers of fecal indicator bacteria are discovered in an environment, it is assumed there is an increased likelihood of pathogens present in the water as well.

Unfortunately, some pathogenic bacteria, viruses, and protozoa may have special survival mechanisms, such as the cyst formation that occurs in Cryptosporidium or the attachment of some viruses to silt or other particles. This means that waters free of fecal indicator bacteria may still harbor or shield these microorganisms on occasion.

This is often true of water which has already undergone treatment for drinking water purposes. Groundwater has traditionally been considered as the potable water source with the least susceptibility to contamination by indicator bacteria or human pathogens such as viruses and cysts—and this is typically true of groundwater from deep and confined aquifers.

Figure 4. Cryptosporidium parvum cysts.

Should fecal indicator bacteria or pathogens commonly associated with humans be found to be present in groundwater in measurable numbers, most likely a nearby hydraulic connection with a contaminated surface water or surface-influenced environment exists. This can be seepage occurring from a wastewater lagoon; contaminated surface water or runoff; a nearby subsurface source of contamination such as from a septic tank or drain field; a broken or leaking sewer line; or an old or improperly designed or operated waste landfill in close proximity to a well or recharge area.

It is important to recognize that while we now know more about bacteria, viruses, protozoa, and other microorganisms than ever before, we still know relatively little about their specific types, activities, and habitats, particularly those that exist in groundwater.

The constant discovery of new pathogens—especially waterborne pathogens—and the association of common bacteria, viruses, or protozoa with specific diseases occurs on a relatively frequent basis. The recent discovery, spread, and resulting pandemic of the new, novel coronavirus underscores this fact.

Test Methods for Waterborne Pathogens

Bacteria, viruses, and protozoa are difficult to count, and most microbiologists believe we have identified fewer than 15% of the bacteria types present in nature.

Figure 5. Common types of waterborne pathogens.

The microbiological quality of groundwater is typically conducted by examining the bacterial presence in water by using the coliform group of bacteria. As previously indicated, most bacteria in the coliform group are generally non-pathogenic in nature, but their presence is believed to provide a probable indication that other potentially harmful bacteria may also be present. This is the reason this group is known as an “indicator group.”

If initial coliform test results are deemed positive, a further examination to verify or refute the presence of Escherichia coliform (E. coli) or fecal coliform bacteria is then performed.

This examination is currently performed through two distinct types of coliform group bacteria tests: presumptive (presence/absence) and colony count (membrane).

Microbiological testing was formerly conducted using the Most Probable Number (MPN) or multiple-tube method where sample water would be placed into five separate test tubes and then seeded with a substance that would result in visible off-gassing and growth within the tube over 24-48 hours if the results were positive for bacteria.

When the initial results were positive, the test would generally undergo a second procedure to confirm the results before final reporting. This type of test was accurate and helped provide a measure of contamination based on the degree and speed of growth and the number of tubes impacted.

In response to U.S. Environmental Protection Agency and industry action, this method was abandoned in favor of the current bacteria test, known as the presumptive or positive/negative test, which is deemed more reliable. This type of test usually requires a total incubation period of 48 hours for accuracy. However, interfering substances in the natural water or improper collection methods have been known to result in false-positive or even false-negative results.

A recent addition, a Colilert test, is a simpler method for rapid results in 24 hours, but the same concerns for interference and wrong results have still been noted.

The final type of microbiological examination is a colony count, which is usually reported in values of Colony Forming Units per milliliter (CFU/mL) or the number of colonies on a plate multiplied by 100 and then divided by the sample volume in milliliters.

Also known as a membrane test, it is likely the most accurate method, although the most expensive and time consuming. With this method, sample water is filtered and collected through an ultra-tight membrane, which is then microscopically examined for bacteria. If any are present, the lab technician counts the number of bacteria colonies that exist across a specific area and provides the results as a number or colony count.

This method also provides a measure of the scale of bacterial contamination and is less likely to be affected by interfering substances or lab error, provided the examiner is qualified and the proper protocol is followed.

Although viruses are just as likely to be present in a contaminated water supply as bacteria, there is currently no presumptive test for viruses. As with the membrane test, the possible presence of viruses in water is performed through microscopic examination, which can also be helpful in identifying the type or strain of viruses present.

Deactivation of microorganisms generally consists of a process of disinfection preceded or followed in many cases by filtration.

Pathogenic bacteria and viruses are usually adequately deactivated through disinfection practices alone such as chlorination, chloramines, ozone, or ultraviolet light. Protozoans, due to their thicker cell walls and resistance to common disinfection processes, generally require supplemental filtration and enhanced disinfection levels and contact time for effective removal or deactivation.

Heterotrophic Group of Bacteria

Examined through use of a Heterotrophic Plate Count (HPC), the heterotrophic group of bacteria is an important topic and their laboratory examination in groundwater chemistry deserves separate discussion.

Heterotrophs describe a group of microorganisms, generally bacteria, that use organic carbon sources to develop and thrive and are found in all types of water and soil. In fact, most bacteria found in drinking water systems are heterotrophs.

The HPC plate count is a method that measures colony formation on culture media of heterotrophic bacteria in drinking water. Thus, the HPC test, also known as a Standard Plate Count, can be used to measure the overall bacteriological quality of drinking water in public, semi-public, and private water systems.

HPC results by themselves are not an indicator of actual water quality or safety, and therefore should not normally be used as either proof of or refuting a potentially adverse human health impact. However, in certain cases, heterotrophic bacteria may be considered as opportunistic bacteria that can affect immune-suppressed individuals, such as HIV or AIDS patients.

The World Health Organization and EPA firmly state that other testing methods, specifically coliform bacteria testing, are better and more reliable indicators than HPC to determine the sanitary conditions of water.

Even though the HPC method does not reveal the specific heterotrophic bacteria present or their sources, it does indicate the organisms that can be cultured or grown are present, which could be as low as 1% of the total bacteria present.

There are several factors that affect the kinds of bacteria and their level of presence shown by an HPC examination. These factors include the type of medium used to grow the bacteria, the temperature used for incubation, how long the plates are incubated, where the water sample was collected, time of year, and age of the sample.

It is also important to note the concentrations and types of bacteria that are demonstrated at the same sampling location can vary over time. Typically, high levels of HPC bacteria in a well, water distribution, or plumbing system result from the regrowth of this bacteria within the system where bacteria that may have resisted the initial treatment continue to grow or those that were injured during the initial treatment sufficiently recovered.

Although there is no established maximum level of HPC bacteria, a lower concentration of heterotrophic bacteria in a water well or finished drinking water is linked to better maintenance of the well, treatment, and distribution systems.

According to this tenet, water treatment techniques should work toward controlling HPC concentrations in surface water and groundwater influenced by surface waters to less than 500 CFU/mL (using standard methods).

Even though this is not a health-based standard, it does reflect the concern that HPC concentrations above 500 CFU/mL can interfere with total coliform and E. coli tests. Generally, even in treated and finished water, a background level of heterotrophic bacteria remains, usually within a range between 10-50 CFU/mL but potentially as high as 100 CFU/mL.

Although an HPC count is often helpful to gauge the current health of a well or water system, of more relative and immediate importance is any sudden or gradual increase in the HPC count from recent, past, or historical test results. This can often be an indication that rapid bacterial growth is occurring within the examined system, necessitating consideration of well or water system rehabilitation, maintenance, or cleaning procedures.

This is a fundamental reason why an HPC test should be conducted on a new or rehabilitated well or water system and then routinely repeated at frequencies or intervals determined by the type of water system and background water quality. The initial test will provide a baseline to evaluate future results.

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This is an abbreviated outline of the most common types of waterborne pathogens, test methods, and possible sources and treatment methods. Additional information is available from numerous sources to provide the required guidance to identify and resolve most problems with waterborne pathogens.

Next month, we will divert from the serious nature of this topic a little to discuss a somewhat lighter subject.

Until then, work safe and smart.


Ed Butts, PE, CPI, is the chief engineer at 4B Engineering & Consulting, Salem, Oregon. He has more than 40 years of experience in the water well business, specializing in engineering and business management. He can be reached at epbpe@juno.com.