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In this month’s column, we offer a consensus review of the NSF International and World Health Organization (WHO) International Symposium on HPC Bacteria in Drinking Water in Geneva, Switzerland, April 22-24.
Heterotrophic plate count (HPC) is a non-specific term for the measure of growth of viable, naturally occurring bacteria in water. The term may encompass bacterial strains of Arthrobacter, Aeromonas, Alcaligenes, Chromobacterium, Pseudomonas, Sarcina, Micrococcus, Flavobacterium, Proteus, Bacillus and others. HPC has been used to evaluate overall finished water quality, maintenance of disinfection residuals, absence of bacterial re-growth and general water treatment effectiveness. In addition, a few groups at the conference suggested high levels of heterotrophic bacteria could be a health risk and should be limited. The debate over the health implications of these bacteria in drinking water ensued in the water treatment industry, mainly due to a misunderstanding of research data and the development of non-health based standards and recommendations to limit HPC bacteria.
The goal of the conference, which was presented by the NSF/WHO Collaborating Centre for Drinking Water Safety and Treatment, was to provide an open forum for international expert debate on the significance and public health implications of HPC bacteria. Following the HPC conference, the WHO held an expert meeting on April 25-26, aimed at arriving at a scientifically based consensus regarding the appropriate interpretations of HPC measurements in drinking water.
The history of HPC bacteria dates back to 1881 when Dr. Robert Koch introduced a method for monitoring water quality by inoculating 1 milliliter (ml) of water to a nutrient gel and incubating for 48 hours at 22oC. At this time, few laboratory methods were known for the growth of bacteria, let alone pathogens. Koch applied his method to the monitoring of source and treated waters but soon recognized the presence of culturable bacteria didn’t necessarily infer the presence of pathogenic organisms. Koch’s bacterial growth methods were used to monitor the public waterworks drawing highly contaminated source water and treating the water with slow sand filtration. When bacterial counts were less than (<) 100 colony-forming units per ml (cfu/ml), epidemics of cholera and typhoid were reduced compared to when counts were greater than (>) 1,000 cfu/ml.
By 1914, the United States was using the total coliform and HPC bacterial tests in combination, with a drinking water standard limiting HPC levels to 100 cfu/ml. After nearly a decade, it was apparent that HPC counts didn’t correlate with bacterial pathogens in water and the standard was abandoned. In 1980, the European Communities issued a guide level for HPC in drinking water of 10 cfu/ml at 37oC and 100 cfu/ml at 22oC. Rapidly replacing reliance of HPC counts as an indicator of water quality was use of indicators (i.e., E. coli) and other fecal bacteria. In 1989, the U.S. Environmental Protection Agency (USEPA) set the level to 500 cfu/ml at 35oC, mainly due to the tendency of high HPC levels to interfere with coliform analysis. Since this time, an alternative to the standard coliform test based on lactose media has been introduced using a defined substrate not impacted by HPC bacteria, i.e., Colilert®, Colisure®.
Detection methods for evaluation of HPC bacteria have evolved over time to improve isolation efficiency. Low nutrient media, such as R2A, is recommended to meet the needs of HPC bacteria, incubated at 28oC for 5-7 days. An incubation temperature of 37oC for 48 hours is also used to include the isolation of HPC bacteria potentially suited for growth in a human host. Isolation values vary dramatically with changes in growth media, temperature and incubation times. Regardless of the method used, only a small fraction (<1 percent) of waterborne microbes are considered culturable HPC bacteria.
Concern over bacteria
Some of the confusion over the significance of HPC bacteria in water results from the fact that waterborne pathogens are almost always heterotrophic; however, isolation of pathogenic microbes usually involves use of specialized growth media and specific growth conditions not found in the general HPC test. To complicate matters further, a literature search of commonly found HPC bacteria, i.e., Pseudomonas, Aeromonas, yields a list of associated illnesses in humans; however, these infections are usually nosocomial, or acquired in the hospital environment, and are usually associated with equipment use or wound infections, not enteric illness. Cases of infection due to HPC bacteria outside of hospitals are very rare, and even fewer are suggested to be of an oral route of infection. Severely ill hospitalized patients (pre-term babies, intensive care patients and AIDS patients) may be vulnerable to HPC bacteria and should drink water that’s sterile. For the majority of the population, sterile water isn’t necessary or desired. Therefore, it’s essential to separate hospital issues of disease transmission from general drinking water issues.
Interestingly, HPC levels in foods haven’t raised the same concern as in the drinking water industry. It’s not unusual to find very high numbers (>50,000 cfu/gram or ml) of HPC bacteria in a wide variety of foods such as pasteurized milk, cheeses, meats, yogurts and fresh produce. HPC bacteria consumed with food is generally several orders of magnitude larger than those consumed in water. A million HPC bacteria have been documented on a single gram of leaf lettuce and carrot sticks.1
A number of factors are related to bacterial re-growth in water including filtration, temperature, disinfectant type and residual, organic matter present, corrosion control and distribution pipe material. Granular activated carbon (GAC), commonly used in the water treatment industry, is known to concentrate bacteria and their growth substrates. As water passes through the GAC, bacteria may also be released. Point-of-use (POU) treatment devices have been shown to reduce the level of waterborne gastrointestinal disease and remove known microbial pathogens. It’s not uncommon, however, to note a significant increase in HPC bacteria post treatment, possibly several-fold higher than the influent water. Re-growth of bacteria in POU devices occurs frequently during periods of non-use. Reports of high levels of HPC bacteria in purified water fueled the debate of their health significance. Many instances were found, however, where large numbers of HPC bacteria were present with no correlation to increased disease. Some studies even suggested that HPC bacteria could out-compete human pathogens and actually demonstrate a protective effect.2
Several studies have evaluated the virulence factors of HPC bacteria. Virulence is defined as the ability of an organism to cause disease. Analysis of the toxicity factors of HPC bacteria in cell culture and the invasiveness of the bacteria isolated from drinking water on blood media impart very few virulence factors.3 In addition, HPC bacteria didn’t survive well at low pH (i.e., conditions of the human stomach). In fact, only 1-2 percent of HPC bacteria were found to have possible virulence factors, but still weren’t associated with human disease. Also, HPC bacteria weren’t found to be virulent when given to severely immunosuppressed mice. Risk assessment analysis of HPC bacteria in water determined that the risk of colonization from oral ingestion of HPC bacteria was <1/10,000 cfu/ml for a single exposure.4
Several large epidemiological studies were conducted in an attempt to define the public health impact of HPC bacteria in drinking water. The first was conducted in the United States and showed that a large number of households using various GAC POU devices had high HPC levels but no demonstrated health effects.(sup>5 Likewise, a recent study conducted in California attempting to correlate HPC in tap water to gastrointestinal illness provided no such evidence.6
In Canada, 300 families were provided with reverse osmosis (RO) units to remove contaminants from their tap water while 300 families were given tap water alone.7 The individuals using RO units experienced 35 percent less gastrointestinal illness than those drinking tap water. HPC bacteria were, however, isolated from the RO units on several occasions and could be correlated to disease in the family.8 Dr. Pierre Payment, the lead researcher on this study, states that the apparent correlation was due to a few families and could have been due to chance, but the POU industry was concerned about this possible correlation.
In a later study, 9 bottled water purified by RO and ozonation was given to one group of families, water bottled at the water treatment plant was given to a second group, and tap water was given to a third group. The water collected at the treatment plant contained high levels of HPC bacteria (as high as 1,400,000 cfu/ml) within a few days, but individuals consuming water with high levels of bacteria reported less illness than those consuming tap water. In fact, the level of illness in tap water bottled at the treatment plant with high HPC was equivalent to those consuming highly purified bottled water with very low levels of bacteria. Therefore, no epidemiological evidence has supported the theory that high bacterial counts in drinking water contribute to an increase in gastrointestinal illness.
Although there is a lack of health-based justification for regulating the level of HPC bacteria in drinking water, a number of countries have set mandatory limits. The German DIN Standard 19636, for example, was developed for water softeners, limiting HPC to below 100 cfu/ml. A careful review of the HPC research indicates the perception of this limit providing increased public health protection is false. While pathogenic waterborne bacteria are generally heterotrophic (i.e., Salmonella, Shigella, Vibrio, etc.) and several opportunistic pathogens may survive and proliferate following water treatment (i.e., Legionella, Mycobacterium avium complex), the presence of these organisms has not been shown to be represented by or correlated with the HPC test.
In summary, an overwhelming body of evidence from human feed studies, animal studies, epidemiological studies, risk assessment, and virulence data all confer there’s no medical rationale to control HPC concentrations in drinking water. The general consensus at the HPC symposium was that such common bacteria in drinking water wasn’t associated with a significant public health risk. The WHO is expected to release an official review document, compiled at the expert conference following the Geneva event, regarding the health significance of HPC bacteria in drinking water to be available later this year.
1. Wadhwa, S.G., et al., “Comparative microbial character of consumed food and drinking water,” Critical Review in Microbiology (in press), 2002.
2. Camper, A.K., et al., “Growth and persistence of pathogens on granular activated carbon,” Applied Environmental Microbiology, 50: 1178-1382, 1985.
3. Edberg, S.C., et al., “Analysis of cytotoxicity and invasiveness of heterotrophic plate count bacteria (HPC) isolated from drinking water on blood media,” Journal of Applied Microbiology, 82: 455-461, 1997.
4. Rusin, P.A., “Risk assessment of bacterial pathogens in drinking water,” Reviews in Environmental Toxicology, 152: 57-83, 1997.
5. Calderon, R.G., “Bacteria colonizing point of use, granular activated carbon filters and their relationship to human health,” Final report CR-813978-01-0, U.S. Environmental Protection Agency, Cincinnati, Ohio, 1991.
6. Colford, J.M., et al., “Participant blinding and gastrointestinal illness in a randomized, controlled trial of an in-home drinking water intervention,” Emerging Infectious Diseases, 8: 29-36, 2002.
7. Payment, P., et al., “A randomized trial to evaluate the risk of gastrointestinal disease due to the consumption of drinking water meeting currently accepted microbiological standards,” American Journal of Public Health, 81: 703-708, 1991.
8. Payment, P., et al., “Gastrointestinal health effects associated with the consumption of drinking water produced by a point-of-use domestic reverse-osmosis filtration units,” Applied and Environmental Microbiology, 57: 945-948, 1991.
9. Payment, P., et al., “A prospective epidemiological study of gastrointestinal health effects due to the consumption of drinking water,” International Journal of Environmental Health Research, 7: 5-31, 1997.
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.