April 2002: Volume 44, Number 4
Microbial Resistance to Disinfectants
by Kelly A. Reynolds, MSPH, Ph.D.
Disinfectants are often the final stage in a multibarrier drinking water treatment system aimed at preventing exposures to waterborne microbial pathogens. Microbes, however, differ greatly in their sensitivity to disinfectants. Specific protozoa, viruses and bacteria are known to be highly resistant to chemical agents and pose a unique challenge to the water treatment industry.
What’s the difference?
Often, the terms sanitized, disinfected and sterilized are used interchangeably when, in fact, they indicate a specific level of activity. In general, a sanitizer is a chemical agent capable of reducing the number of microbes to a safe level. This level has been defined as a 3-5 log reduction (99.9 percent to 99.999 percent) of disease causing bacteria within a population, or a specific test population, respectively. The allowable kill time to determine the efficacy of a sanitizer is 30 seconds or less.
A chemical disinfectant, also a chemical agent, must destroy at least 99.999 percent of bacterial pathogens within 5-10 minutes. Although not necessarily able to kill spore-forming bacteria or all viral pathogens, disinfectants are required to kill more pathogenic bacteria than a sanitizer. In addition, disinfectants are not recommended for use on human skin, but rather for environmental surfaces or water.
Confused? It gets worse. Iodine, for example, is a sanitizer at 25 parts per million (ppm) but a disinfectant at 75 ppm. Thus, how a chemical is classified is largely based on specific use concentrations. Other examples in which the use concentration of the product defines its classification include chlorine and quaternary ammonium compounds (quats), which are found in many household disinfectants.
Other chemical agents, known as sterilants, render the exposed environment completely free of all microbial life, including the highly resistant spores (see Table 1).
Glutaraldehyde and formaldehyde are examples of chemicals that can sterilize the exposed environment. Certain aldehydes have been identified as possible carcinogens and thus have limited use. Sterilization can also be achieved by physical conditions of pressure and heat, known as autoclaving. Autoclaves are often used in the clinical arena to ensure sterile utensils, without the use of potentially toxic chemicals.
Additional methods for controlling microbial agents in food and water include boiling, irradiation and pasteurization. Boiling water at 100oC for 30 minutes kills most microbial pathogens and vegetative forms of bacteria but may not kill bacterial endospores. Three 30-minute intervals of boiling at 100oC followed by cooling kills the bacterial endospores. Irradiation involves the use of microwaves, UV, gamma rays or x-rays to inactivate microorganisms. Applied to foods, irradiation can increase the shelf life of fruits and vegetables by up to 500 percent. This technology has also been applied successfully to drinking water. Pasteurization uses mild heat to reduce the number of microbes in a product, i.e., milk. Milk is typically pasteurized by heating it at 63oC for 30 minutes.
How do they work?
Chemical agents used as germicides destroy microbial pathogens using a variety of biochemical mechanisms, including cell membrane destruction, interference of vital mechanisms, or inactivation of critical processes. Oxidizing agents -- such as ozone and chlorine -- commonly used for drinking water treatment, typically destroy the outer membrane of the bacterial cell and render it noninfectious. Free chlorine has been the choice disinfectant for municipal water treatment for nearly 100 years. The oxidizer is highly effective against most bacteria but only moderately effective against spores. Chlorine compounds are inexpensive, rapid and effective disinfectants, but can be limited by poor water quality parameters such as organic matter, and are thus used primarily in the end treatment step. Chlorine does carry a residual and thus provides additional treatment benefits within distribution systems.
Quats, commonly used for surface decontamination, tend to block the uptake of nutrients into the cell and prevent excretion of waste products. Both of these actions have deadly effects on microbes.
There’s some evidence sanitized environments may leave a subset of the original bacterial population that’s resistant, allowing this “superbug” population to repopulate the surface. Concern has been voiced regarding the overuse of sanitizers. The solution to this occurrence is usually to shock the surface with adequate disinfectant concentrations since disinfectants are more powerful than sanitizers. Pathogen resistance to chemical disinfectants is typically an inherent characteristic and not one that’s acquired or initiated due to exposure to disinfectants.
Mechanisms of resistance
Microorganisms have evolved with specialized structures aiding in their survival. Certain bacteria produce a gelatenous material known as exopoly-saccharide to form a biofilm. Biofilms help organisms stick to environmental surfaces and physically protect them from exposure to harmful disinfectants or other detrimental environmental conditions. This slimy substance may be 100 or more times the mass of the bacterial cells. While the bacteria that initiate biofilms are often not harmful, pathogenic bacteria may also stick to the biofilm and share the protective nature of the environment. Viruses associated with biofilms can survive 2-10 times longer than free-floating populations. Biofilm-associated bacteria have been reported as being up to 3,000 times more resistant to free chlorine, compared to their free-floating counterparts.1
Another survival mechanism of bacteria is the formation of spores. Vegetative cells become spores when they’re deprived of essential growth nutrients. Formed only under conditions of stress and in Gram positive strains, bacterial spores -- or endospores -- can survive extreme heat, cold, drying and chemical exposures. Indicative of spores is the production of specific spore proteins and the lack of proteins known to occur in the vegetative state. Under more favorable conditions of moisture and temperature, endospores can revert from their dormant state back to actively reproducing vegetative cells. The best known spore-forming bacteria are Bacillus (genus of anthrax producing bacteria) and Clostridium spp. (genus of botulism producing bacteria). Many endospores are capable of survival in their dormant state for years (some having been shown to survive for thousands of years).
The power to resist
Other bacteria resistant to disinfectants include Mycobacterium avium, Legionella, and Staphylococcus aureus. Environmental isolates of M. avium, an opportunistic pathogen commonly found in water and soil, are known to be highly resistant to disinfectants including chlorine, monochloramine, chlorine dioxide and ozone. M. avium strains are up to 2,300 times more resistant to chlorine than E. coli, an indicator of drinking water quality. Levels of chemical disinfectant used in drinking water treatment are unlikely to be effective against many Mycobateria species. 2
As part of their life cycle, protozoa often have an environmentally resistant stage. Cryptosporidium, for example, forms an oocyst that aids in its environmental survival. Although susceptible to freezing and drying, oocysts can remain infectious for more than a year in aquatic environments. Largely unaffected by chlorine, water treatement facilities rely on filtration technology to remove these small protozoa (6-8 m). Past treatment failures have correlated to outbreaks from Cryptosporidium, leading to significant morbidity and mortality. For this reason, severely immunocompromised individuals are advised to boil their water for at least one minute prior to consumption, primarily to reduce the risk of cryptosporidiosis.
The Norwalk virus is thought to be one of the primary agents of viral waterborne outbreaks in the United States. The increased resistance of the Norwalk virus to chlorine disinfectant may be the cause of significant morbidity in the United States each year. Researchers found that 3.75 ppm of chlorine was effective against poliovirus and rotavirus but did not inactivate the Norwalk virus.3 It remained infectious for five out of eight volunteers. Even after exposure to 10 ppm of chlorine, it caused illness in one volunteer.
The increased emergence of antibiotic resistant bacteria suggests the need for heavier reliance on disinfection practices to prevent initial infection. Biocidal agents used in water treatment have been essential for virtually eliminating once rampant diseases like cholera, typhoid and dysentery. In developing countries where disinfectants aren’t used in water treatment, these diseases are still responsible for significant numbers of fatalities and illnesses.
The ideal disinfectant hasn’t yet been developed. The variable nature of microbial populations and their ecological habitat alter the efficacy and predictability of the disinfection process. The innate resistance of the microbe and the intrinsic power of the disinfectant must be in balance, keeping in mind the presence of organic matter, turbidity, excessive numbers of organisms, exposure times or dilution use concentrations, pH, temperature, and water hardness may affect treatment. Even well operated water treatment plants cannot ensure drinking water will be completely free of harmful microbes such as Cryptosporidium.
Water can be filtered to remove Cryptosporidium oocysts and the cysts of another protozoan parasite, Giardia lamblia. Reverse osmosis membranes and other point-of-use filters with an “absolute” (not “nominal”) pore size of one micron or smaller will remove oocysts (viruses, however, can pass through a one-micron filter). The pore size of microfiltration is still too small for oocysts to pass through. Boiling water for at least one minute is the recommended treatment for severely immunocompromised individuals.
1. LeChevallier, M.W., et al., “Inactivation of biofilm bacteria,” Applied and Environmental Microbiology, 54(10): 2492-9, 1998.
2. Keswick, B.H., et al., “Inactivation of Norwalk virus in drinking water by chlorine,” Applied and Environmental Microbiology, 1985.
3. Taylor, R.H., et al., “Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium,” Applied and Environmental Microbiology, 66(4):1702-5, April 2000.
About the author
Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on development of rapid methods for detecting human pathogenic viruses in drinking water. She holds a master of science degree in public health (MSPH) from the University of South Florida and doctorate in microbiology from the University of Arizona. Reynolds also has been a member of the WC&P Technical Review Committee since 1997.
Table 1. Microbes ranked in order of decreasing resistance to disinfectants
* Mycobacterium tuberculosis
* Non-lipid viruses (i.e., hepatitis A, poliovirus)
* Vegetative bacteria
* Lipid viruses (i.e., HIV)