http://informahealthcare.com/mby ISSN: 1040-841X (print), 1549-7828 (electronic) Crit Rev Microbiol, Early Online: 1–8 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2013.849655
REVIEW ARTICLE
Drinking water microbial myths* Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Martin J. Allen1, Stephen C. Edberg2, Jennifer L. Clancy3, and Steve E. Hrudey4 1
Water Research Foundation (retired), Denver, CO, USA, 2School of Medicine, Yale University, New Haven, CT, USA, 3J. Clancy & Associates, St. Albans, VT, USA, and 4Environmental & Analytical Toxicology, University of Alberta, Edmonton, AB, Canada
Abstract
Keywords
Accounts of drinking water-borne disease outbreaks have always captured the interest of the public, elected and health officials, and the media. During the twentieth century, the drinking water community and public health organizations have endeavored to craft regulations and guidelines on treatment and management practices that reduce risks from drinking water, specifically human pathogens. During this period there also evolved misunderstandings as to potential health risk associated with microorganisms that may be present in drinking waters. These misunderstanding or ‘‘myths’’ have led to confusion among the many stakeholders. The purpose of this article is to provide a scientific- and clinically-based discussion of these ‘‘myths’’ and recommendations for better ensuring the microbial safety of drinking water and valid public health decisions.
Drinking water, microbial monitoring, pathogens, public health decisions, waterborne disease
Introduction The provision of a safe and sustainable drinking water supply is one of the hallmarks of a successful society. Without safe drinking water, public health is always at risk and the economic potential of the community cannot be realized. In 1849, Dr. John Snow demonstrated that cholera was attributed to drinking water contaminated by microbial pathogens by dispelling the prevailing myths of his time that cholera was caused by ‘‘odorous vapours’’ (VintenJohansen et al., 2003). Since then, scientists, engineers and public health officials have endeavored to determine which water treatment processes will remove or inactivate human pathogens in drinking water, and how best to monitor these processes and the water distributed to consumers. This article describes: the chronology of microbial monitoring practices thought to best ensure safe drinking water; key references that support or refute current monitoring approaches; discussion of the ‘‘myths’’ that have evolved and continue to be perpetuated contrary to the published science; and how these ‘‘myths’’ can hamper making correct and timely public health decisions. Drinking water microbial ‘‘myths’’ include: Routine pathogen monitoring is a credible tool to identify potential health risks;
Address for correspondence: Martin J. Allen, Water Research Foundation (retired), 6666 West Quincy Ave, Denver, CO, USA. Tel: 303-9066951. E-mail: [email protected] *The authors dedicate this paper to Dr. Donald J. Reasoner, Chief – Microbial Contaminants Branch at the USEPA, inventor of R2A HPC media for HPC bacteria, recipient of numerous awards for scientific advances in drinking water microbiology, and a fellow ‘‘myth buster’’.
History Received 24 April 2013 Revised 24 September 2013 Accepted 25 September 2013 Published online 25 November 2013
The presence of coliforms or fecal coliforms always signifies an elevated health risk; Enumeration of indicator bacteria in treated drinking water is necessary to identify potential health risk; Certain microorganisms when found in drinking water pose an increased health risk; The ‘‘very young’’ and ‘‘elderly’’ are often considered ‘‘immunocompromised’’; Molecular microbial methods will soon offer the promise of rapid detection of pathogens to make correct and timely public health decisions; Only certified laboratories are able to accurately analyze for coliforms and E. coli; and Prescribed regulatory microbial monitoring frequencies are sufficient to correctly assess health risk. Key references with in-depth data/discussion provide a broader understanding as to the origin of these myths.
Evolution of indicators to protect public health As early as the late 1800s, it was realized that monitoring for all known human pathogens (many pathogens were unknown at that time, e.g. viruses, Legionella, Cryptosporidium), was impossible and that an alternative approach for routine monitoring of the microbial safety of drinking water was necessary. Frankland & Frankland (1894) stated ‘‘The longer the bacteriological examination of water is practiced the more evident does it become that in searching for pathogenic organisms special methods must be devised and adopted according to the nature of the particular microbe of which we are in quest, and that only any possibility of such pathogenic bacteria being found in the course of ordinary plate cultivations made with natural waters, the colonies of the common
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
2
M. J. Allen et al.
water-bacteria almost invariably so predominating as to exclude all others present in small numbers’’. It was also noted the need for ‘‘ . . . larger volumes of water, as in this manner, the chance of discovery are correspondingly increased’’. And still today, pathogen monitoring of drinking water to make timely and correct public health decisions is not realistic, although some continue to believe that such monitoring may be possible. Allen et al. (2000), Payment & Pintar (2006), and Rizak & Hrudey (2006, 2007) described the major issues that make pathogen monitoring in drinking water impractical or currently ineffective for protecting public health. These include: Diversity of human pathogens, i.e. protozoa, bacteria, viruses, that may be present in drinking water, and therefore the multitude of complex standardized methods that would be necessary to successfully monitor for each type of pathogen. The extremely low numbers of pathogens in surface waters and wastewaters in comparison to the entire total microbial flora in these waters. The lack of standard methods that provide credible data, i.e. specificity, sensitivity and reproducibility, to validate the effectiveness of the in-place water treatment process and to make correct and timely public health decisions. The inability of methods to determine whether pathogens are infectious, viable, or human or non-human infectious strains. The need to examine large volumes (41.0–100 L) of water to have any reasonable chance of detecting the target pathogen. The de-sensitization of any method involved with concentrating large volumes for subsequent analysis. Very few water laboratories have resources (equipment, biohazard-rated facilities and qualified analysts) capable of performing wide-spectrum pathogen monitoring. Lack of organizations qualified and with the on-going resources to certify pathogen methods, and lack of standards. The days to weeks required to obtain a test result with sufficient accuracy to make informed public health decisions based on the monitoring data. Positive Predictive Values (PPV), the probability that a given pathogen ‘‘detection’’ is accurate for routine treated water quality monitoring for pathogens is inevitably unacceptably low. The concept of PPV (Hrudey & Leiss, 2003; Hrudey & Rizak, 2004) assesses the credibility of health risk data as it relates to the accuracy of analytical data with the method used and the probability the target contaminant (biological or nonbiological) is present frequently enough for the analytical method to provide accurate detection. All analytical methods and those especially used for detection of pathogens have significant false positive and false negative rates that compromise the credibility of the data to make correct public health decisions. Furthermore, such methods do not always determine viability, infectivity and human versus non-human strains. In the case of treated drinking water, where occurrence of pathogens should be rare, most samples tested will be truly negative, but monitoring predominately negative samples
Crit Rev Microbiol, Early Online: 1–8
with a method that has a non-zero false positive rate assures that a substantial number of false positives will occur. The rarer the occurrence of pathogens and/or the higher the false positive rate, the larger will be the number of false positives relative to the rare true positives. This inevitable reality with false positives exceeding true positives means the PPV will be much less than 50% (equal likelihood of a correct or erroneous result). For pathogen monitoring in treated drinking water, the PPV will commonly be much less than 10%, less than one chance in 10 of the result being true (Rizak & Hrudey, 2007). With all the above noted barriers to credible pathogen monitoring known, although many are dismissed even today, indicator or surrogate bacteria are used to signal recent fecal contamination and thus the possible presence of pathogens. (Edberg et al., 2000; Rochelle & Clancy, 2006). The criteria of a suitable sanitary bacterial indicator include: Always present in animal and human feces; Present in high numbers for higher probability of detection; Persistence in the environment/drinking water similar to that of pathogens; Does not multiply in the environment; and Simple, rapid, accurate, and inexpensive methods are available. Today the most widely used indicators for drinking water safety and quality include: Total Coliforms Fecal Coliforms – the correct term is ‘‘thermotolerant coliforms’’ Escherichia coli Heterotrophic Plate Count (HPC) bacteria Biological indicators other than E. coli including Enterococcus (or fecal Streptococcus), Clostridium perfringens spores, somatic coliphages and male-specific coliphages have been suggested, but may not be suitable or practical for routine monitoring of drinking water (Edberg et al., 2000), i.e. complex methods, methods not standardized, data interpretation (does the mere presence of these microorganism constitute a true health threat from consumption of drinking water?). The functional definition of total coliforms in Standard Methods for the Examination of Water and Wastewater, 22nd edition (APHA, AWWA, WEF, 2012) is the ability to metabolize lactose to produce acid and gas, or possession of the enzyme b-galactosidase (only E. coli possesses b-glucuronidase). The coliform genera and their sources are: Escherichia – human and animal feces (predominant facultative anaerobic bacterium in the colon of all warm-blooded mammals; 108–109/g in feces); Enterobacter – predominately environment, little in feces; Klebsiella – predominately environment, little in feces; Citrobacter – environment; and Serratia – environment. Analysis for total coliforms in drinking water can be performed per Standard Methods using: (1) Multiple Tube Fermentation (MTF) method; (2) Membrane Filtration (MF) method, and (3) Chromogenic/Enzymatic (CE) method. Of these methods, the MTF method is the oldest and, for drinking water, has been largely replaced by either the MF or CE
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
DOI: 10.3109/1040841X.2013.849655
Drinking water microbial myths
3
Figure 1. Scanning electron micrographs (SEM) showing bacteria colonizing a water main tubercles – New Haven, CT (Allen et al., 1980).
methods since the MTF method is labor-intense, timeconsuming and does not provide better data (see section 9221C, Standard Methods, Table 9221:II, Table 9221:III, Table 9221:IV – 95% Confidence Limits). Also the adoption of the Presence/Absence concept by many national health organizations eliminates the need for enumeration data (see later discussion). For drinking water analysis, it is common practice to use the membrane filter technique (Standard Methods-9222B) using LES Endo agar. Further in 9222B-4f, coliform verification is recommended since ‘‘typical sheen colonies may be produced occasionally by noncoliform organisms’’. Also ‘‘Atypical colonies (dark red or nucleated colonies without sheen) occasionally may be coliforms. Preferably, verify all typical atypical colony types, but at a minimum, verify all typical and five atypical colonies per membrane’’. Further, ‘‘For drinking water, verify all colonies on Endo media by swabbing the entire membrane or picking at least five typical colonies and five atypical colonies from a given filter culture. Verification methods are described in 9222B-4f require additional time (up to 48 h). The requirement to verify ‘‘typical and atypical’’ coliforms colonies from drinking water samples may not be routinely practiced with health advisories issued without verified microbial data. It is ill-advised and could be considered irresponsible to issue health advisories without conclusive microbial evidence of elevated risk or other risk factors, e.g. chlorination failure, water quality indicators, unexplained disease. The MF method is often used because it has been assumed that enumeration, i.e. number of colonies on a membrane provides data to ensure correct public health decisions. However, the time required (48 hr) to complete verification per Standard Methods when using the MF method greatly compromises timely and correct public health decisions. In contrast, the enzymatic (chromogenic) methods for coliforms and E. coli, described later and included in Standard Methods, provide verified data in 24 hr or less.
Heterotrophic bacteria and the coliform genera Enterobacter, Klebsiella and Serratia are known to grow post-treatment in the water distribution networks (Allen et al., 1980; Edberg et al., 1994a) when there are sufficient nutrients, e.g. 410 mg/L of assimilable organic carbon (AOC) for unchlorinated systems and 4100 mg/L AOC for chlorinedisinfected systems; warmer water temperatures (415 C) often found during summer months; long residence time of the water within the distribution system/storage reservoirs (days to weeks); and low disinfectant residuals especially at the far reaches of the distribution system (Figure 1a and b). Thus, total coliforms cannot be relied upon as an indicator of fecal contamination (elevated health risk) and the mere presence of total coliforms should not be used to trigger a health advisory. The fecal coliform method with higher temperature incubation of cultures was thought to favor the growth of only fecal-associated E. coli and inhibit growth of the other four coliform genera. This assumption was incorrect since up to 15% of Klebsiella (non-fecal origin) can grow at 44.5 C and up to 10% of E. coli are unable to grow at 44.5 C, thus a potential of a 25% error (false positive or false negative) (Edberg et al., 1994b). It was found that fecal coliform or more appropriately ‘‘thermotolerant coliform’’ was a poor surrogate for E. coli and has subsequently been removed from drinking water regulations. The same sciencebased rationale is appropriate for elimination of the use of the fecal coliform method for determining waste water quality. As shown above, only E. coli is extensively found in fecal matter and thus best supports its logical preference as an indicator of the possible presence of pathogens in drinking water. Tables 1 and 2 support the relevance of E.coli as the best indicator of fecal origin. A much earlier study (Iowa State College, 1921) reported the percentages of fecal types of coliform bacteria as 94.1% in human feces and 92.6% in animal feces. In both studies (Iowa State College, 1921 and Dufour, 1977) spanning 56 years, the percentage of the E. coli populations among coliform bacteria in fecal matter is consistently greater than
4
M. J. Allen et al.
Crit Rev Microbiol, Early Online: 1–8
Table 1. Relative number of fecal and nonfecal types of coliform bacteria in various substances.
Source observed Human feces Animal feces Water Milk Grain Soil
Table 3. Bacterial populations in uncooked vegetables log/gram (total coliforms, fecal coliforms, HPC bacteria). Vegetable
Number of strains
Percentage Aerobacter aerogenes*
Percentage Escherichia coli
2534 1832 2137 1382 288 853
5.9 7.4 35.2 43.1 81.7 88.1
94.1 92.6 64.8 56.9 18.3 11.9
Alfalfa sprouts Broccoli Cabbage Carrots Cauliflower
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Celery
*Renamed Enterobacter aerogenes Source: Iowa State College (1921).
Cucumber Lettuce
Table 2. Percentages of coliform genera in human and animal feces (Dufour, 1977). Mixed Salad
Animal (Number Examined)
E.coli (%)
Klebsiella spp. (%)
Enterobacter/ Citrobacter (%)
Chicken (11) Cow (15) Sheep (10) Goat (8) Pig (15) Dog (7) Cat (7) Horse (26) Human (26) Average
90 99.9 97 92 83.5 91 100 100 96.8 94.5
1 – – 8 6.8 – – – 1.5
9 0.1 3 – 9.7 – – – 1.7
Radish Spinach Tomato
# Tested 2 3 1 36 8 12 1 9 36 2 4 4 8 4 7 8 36 1 3 12 8 16 8 36
TC
ND ND 44.0 0.6–43.0 ND ND 6.3 ND ND 2.4 ND ND ND ND ND ND 4.9 ND ND ND ND ND 5.2 ND ND 4.2 ND ND ND ND ND 5.4 5.4 ND ND ND 6.1 5.5 6.2 ND ND 5.6 ND ND ND 2.6 5.2 ND
Irrelevance of enumerating microbes for drinking water safety The membrane filtration (MF) method has been the preferred method for decades since it was thought to accurately enumerate coliforms, fecal coliforms and E. coli. This assumption is not supported from statistical data although many microbiologists, engineers, public health officials, etc. remain unconvinced, i.e. enumeration is needed to make
HPC
Reference
8.2 8.8 4.5 6.6 ND 5.8 7.3 4.8 5.2 6.0 4.5 5.9 ND 5.2 6.2 ND 5.7 5.9 7.1 8.3 ND 5.9 ND 5.5
Callister & Agger (1987) Reina et al. (1995) Callister & Agger (1987) Albrecht et al. (1995) Monge & Chinchilla (1996) Garg et al. (1990) Beuchat & Brackett (1990) Garg et al. (1990) Albrecht et al. (1995) Callister & Agger (1987) Garg et al. (1990) Reina et al. (1995) Monge & Chinchilla (1996) Garg et al. (1990) Callister & Agger (1987) Monge & Chinchilla (1996) Albrecht et al. (1995) Garg et al. (1990) Vescovo et al. (1995) Beuchat & Brackett (1990) Monge & Chinchilla (1996) Garg et al. (1990) Monge & Chincilla (1996) Albrecht et al. (1995)
ND, not determined. *Provided by Dr. Eugene W. Rice, USEPA.
Table 4. 95% Confidence limits (CL) based on colony-forming units (CFU) for membrane filtration (MF) results using 100 mL samples (abridged from Standard Methods – Table 9222 III). Number of CFUs counted
93%. In particular, the 1977 data (Dufour, 1977) shows the percentage of Klebsiella spp. and Enterobacter/Citrobacter spp. among coliform bacteria in fecal matter is much lower than E. coli, making these species poor indicators of fecal contamination. Despite the overwhelming evidence that E. coli is the best indicator of potential health risk, i.e. possible presence of pathogens, total coliform monitoring for drinking water continues to be considered as a suitable indicator of possible health risks by some health agencies and public health officials. While historically the safety of drinking water has focused on the presence of total coliforms and fecal coliforms and not total bacteria, foods, in contrast, routinely contain high populations of bacteria. All foods contain very high numbers and a wide spectrum of microorganisms that rarely pose a health risk (Wadhwa et al., 2002). Coliforms, fecal coliforms and heterotrophic plate count (HPC organisms) are not unique to drinking water but are the normal flora found in the environment including vegetables that are consumed uncooked without adverse health effects. Table 3 provides such information.
FC
4 10* 20
Lower CL
Upper CL
1.0 4.7 12.2
10.2 18.4 30.8
*Ten colony forming units (CFUs) have a confidence range of 4.7–18.4 CFUs.
correct public health decisions; Table 4 shows the confidence limits of the MF method. Studies (Brenner & Rankin, 1990; Burlingame et al., 1984; Elmund et al., 1999; Jacobs et al., 1986; Rice et al., 1987; Rice et al., 1989) showed membrane filtration methods often recovered fewer target organisms than liquid methods for a variety of reasons including flawed membranes (pores too small/large, inhibitory residues); other high HPC bacteria (4500 CFU/mL) in a sample can desensitize lactose-based method (false negatives); and liquid-based methods recovery, i.e. enzymatic (chromogenic) is generally higher than MF methods (Edberg, 1994b). With the known inherent statistical variability of microbes in water samples shown in Figure 2 (Christian & Pipes, 1983), the United States Environmental Protection Agency (USEPA, 1989) adopted the presence/absence (P/A) concept for compliance monitoring as have other equivalent health organizations. In Figure 2, in a random dispersion of thirtysix 100-mL samples of water containing coliforms, the probability of one 100-mL sample having bacteria was: 37%, one bacterium; 37%, no bacteria; 18%, two bacteria and 7%, 42 bacteria. As bacteria are particulates, they are not uniformly distributed (randomness), and the enumeration of bacteria cannot be related to level of health risk. As such the
DOI: 10.3109/1040841X.2013.849655
Drinking water microbial myths
5
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Figure 2. Variability in bacterial counts detected in 100 mL grab samples from a well-mixed drinking water bulk sample (Christian & Pipes, 1983).
concept of P/A has been adopted as the science-based approach for assessing risk, thus questioning the practice of enumeration of indicator bacteria by the MF technique with its flaws noted above to be able to make a more accurate determination of potential health risk.
Public health relevance of coliforms and non-coliforms in drinking water Based on the literature for assessing the safety of drinking water, the following conclusions are made: Only the presence of E. coli in drinking water, as an indicator of fecal contamination, has potential public health significance (Edberg et al, 2000); USEPA uses E. coli only to assess potential health risk in the Revised Total Coliform Rule in 2016 (Federal Register, 78 (30), Feb 13, 2013). Total coliforms will be used as a nonhealth based water quality indicator for water treatment and distribution systems. HPC bacteria in drinking water have no public health significance, but can be used to monitor significant water quality changes as drinking water is distributed to consumers. (Allen et al., 2003). Bacteria that may be found in treated drinking water that have no public health significance from ingestion include: Klebsiella (Duncan, 1988); Aeromonas (Edberg et al., 2007); Pseudomonas aeruginosa (Hardalo & Edberg, 1997); Helicobacter (Johnson et al., 1997); HPC bacteria (Allen et al., 2003; Payment et al., 2003). The respiratory illness, Legionnaires’ disease, has often been associated with drinking water as the source of the organism and the bacterium can be isolated from even the most aggressively treated drinking water. It is important to understand the epidemiologicallyproven mode of infection is air-borne, i.e. shower aerosols, not consumption of drinking water (Mulder et al., 1986). Although Legionella can be found occasionally in treated drinking water, it is the improper distribution and storage within buildings and lack of World Health Organization (WHO)-recommended water quality management plans within buildings (hospitals, nursing homes, high rises) that promote growth of Legionella that can result in a higher risk of disease. The similar situation applies to Mycobacterium avium complex – MAC (Whiley et al., 2012) that may be present in drinking water, posing little if any health risk
from ingestion, but may pose greater health risks related to warm-water distribution systems, showers, swimming pools, hot tubs, spas, etc. Fungi are another group of microorganisms present in treated drinking water. They occur naturally in the environment, all surface waters, foods, air and some in treated drinking water (Kelley et al., 2003; Nagy & Olson, 1982, 1985, 1986; UK Department for Environment, Food & Rural Affairs, 2011; UK Water Industry Research Microbiology Database (UKWIR), 2008). These organisms are usually present in low numbers in drinking water distribution systems and are associated with taste and odor complaints, but not a direct health concern. The UKWIR database states ‘‘Effective water treatment and maintaining the integrity of the distribution systems will prevent most micro-fungi and yeast from entering water supplies’’. Further, ‘‘Maintaining an effective disinfectant residual will help to prevent the growth of fungi’’. At this time there is no clinical evidence to suggest that fungi present in drinking water poses a health risk. Immunocompromised populations It is often broadly and incorrectly stated that immunocompromised individuals include the very young/infants and the elderly. The general term immunocompromised is meaningless unless the specific immune defect is known and measureable. Immunocompromised individuals are people with significantly measureable decreases in their immune function and include patients on active anti-cancer drugs, HIV/AIDS or other chemotherapies, plus the undiagnosed, e.g. HIV/AIDS. A hematology profile showing abnormal values for gamma globulins, white blood cells, red blood cells, liver function, etc., identifies an immunocompromised individual. Avoiding consumption of treated drinking water is not necessarily recommended given the natural microflora in foods and the robustness of the host defenses of the gastrointestinal tract (lysozyme, acid pH, proteolytic enzymes, bile, etc.) (Duncan & Edberg, 1995). Individuals who are truly immunocompromised per a medical tenet are aware of their condition and should be advised by their doctor to take appropriate precautions. While the young and the elderly may be sensitive populations showing a higher prevalence of more severe outcomes in some outbreak scenarios, it is not possible to define susceptibility of the
6
M. J. Allen et al.
young or elderly accurately by age. Rather susceptibility must be defined by a physiological state – diabetes, obesity, hypertension, etc. Thus, regulations to minimize health risk from drinking water need to be based on the medically-based science as to specific populations at risk, not generalized, non-definable populations.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Relevance and status of molecular techniques for assessing presence of human pathogens in drinking water For decades the use of molecular methods to rapidly identify human pathogens in drinking water has been thought to be a promising public health approach. Since the 1990s, the Water Research Foundation (www.WaterRF.org) funded 47 projects that involve molecular techniques with a value over $7.8 million (personal communication, John Albert, Water Research Foundation). These projects have touched upon virus, protozoa and bacterial detection in drinking water. Many other research organizations worldwide have also funded projects on developing molecular methods. There is progress in using molecular methods in support of Quantitative Microbial Risk Assessments (determining the adequacy of in-place water treatment processes to remove/ inactivate pathogens from drinking water) and use of quantitative polymerase chain reaction (qPCR) methods as an epidemiological tool (presence of enterococci, Bacteroides, enteric viruses) for recreational waters (Borchardt et al., 2012; Wade et al., 2010; US EPA, 2012b). With regard to drinking water, there are several challenges that need to be addressed and understood when considering the use of molecular methods to assess potential health risks that may be associated with viable pathogens in drinking water. Some critical questions include: Of the array of pathogens which one(s) should be selected to best determine risk? What is an appropriate protocol for analyzing for targeted organisms? Does that presence of microbial nucleic matter (fragments of DNA/RNA) in drinking water pose a health risk, i.e. capable of causing disease? What ‘‘positive’’ result or signal would imply an increased health risk? How would molecular methods be certified/approved based on standardized criteria to ensure sensitivity, specificity, reproducibility and by which certifying organizations? What criteria (appropriate resources, technical staff and methods training/experience) are essential to routinely use molecular methods with confidence? What are the sample preparation and storage issues, e.g. loss of sensitivity during concentration procedures that need to be addressed to ensure credible data? Is the PPV adequate to make credible public health decisions? Two published studies on molecular methods (Hill et al., 2010; Nocker et al., 2010) reported: Water utility laboratory managers view molecular methods favorably with the major benefit being time to detection;
Crit Rev Microbiol, Early Online: 1–8
Despite the favorable impression, many managers stated that their laboratories do not currently have the resources or expertise to use these methods; Researchers and utilities surveyed by the United States Centers for Disease Control and Prevention using molecular methods for drinking water monitoring see routine pathogen screening possible within the next 10 years provided barriers are overcome, e.g. method standardization, sample preparation issues, such as loss of sensitivity during concentration procedures (Hill et al., 2010).
Rapid microbial methods can improve public health protection Beginning in the late 1980s, a new type of microbial analysis drawing from clinical technologies was developed based on CE that simultaneously measure the P/A of both total coliforms and E. coli. The use of CE methods along with sanitary surveys can greatly enhance public health protection, especially for smaller remote communities found in every country; for example 93% in the US and 75% of Canadian public water systems serve 510 000 people (Allen et al., 2000). Several CE methods are approved for detection of total coliforms and E. coli including: ColilertÕ , Colilert-18Õ , ReadycultÕ , E*Colite TestÕ , ColitagÕ , and ColisureÕ . Despite the enormous promise of enhanced public health protection for small to medium-sized water utilities by using CE methods, there are many indefensible and erroneous barriers that prevent their greater use (Allen et al., 2010). These unwarranted barriers to wider adoption include beliefs that: (1) Utility operators do not have ability or training; (2) Operators may ‘‘cheat’’ to ensure negative results; (3) Cost of equipment and media is prohibitively expensive; (4) Certified laboratories may have fewer samples to process with loss of income; (5) The laboratory certification authority will be perceived as having less control; (6) Public health authority could be perceived as abrogating their oversight; and (7) Current regulations require samples be sent to certified laboratories. CE methods are successfully used by small and remote water systems in Alaska and First Nations in Quebec Province of Canada (Allen et al., 2010). The elimination of these barriers, particularly regulatory constraints, would provide a much higher level of public health protection especially for the medium to smaller water systems where access to certified laboratories is severely limited. If there is a genuine interest to protect health protection rather than maintaining the status quo, the use of CE methods on-site by trained operators should be embraced for both large and smaller water utilities.
Unrealsitic expectations for microbial monitoring While the use of E. coli to help determine potential health risk in treated drinking water is useful and probably the best that can be done at this time, in reality, microbial monitoring as now practiced or required is a very imperfect and questionable approach to credible health protection considering: The percentage of drinking water tested for indicator bacteria is 0.0000002–0.0000005% meaning 99.99998–99.99995% is not tested;
Drinking water microbial myths
DOI: 10.3109/1040841X.2013.849655
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
The lack of plausibility in concluding an elevated health risk with a ‘‘positive’’ sample given the miniscule volume of water tested; Understanding that even monitoring using the current best indicator organism (E. coli) cannot be shown to have prevented any drinking water-borne outbreaks (Hrudey et al., 2008) – results are reported after contamination has occurred – but data on such indicators are certainly important for investigating and verifying outbreaks; Under unique situations (water main breaks, crossconnection failures, earthquakes, hurricanes, tsunamis, other catastrophic events), on-site microbial monitoring to assess health risk is appropriate; and, While microbial monitoring is one tool that can be used for assessing water quality and potential health risk, there are more suitable non-microbial water quality and treatment performance parameters (chlorine residual, turbidity) and other approaches, e.g. sanitary surveys, integrity of the water distribution network, that provide a much higher degree of confidence on the safety of drinking water. A more pragmatic and defensible approach for better ensuring safe drinking water should include: Appropriate treatment processes in place as determined by source water quality; Continuous on-line monitoring of the treatment processes that ensures the processes are optimized, e.g. use of on-line turbidimeters, disinfectant residuals at the periphery of the water distribution network. Trained and certified operators with certification to perform microbial analysis; Appropriate scheduled operation and maintenance of the treatment processes; Appropriate disinfection residuals throughout the storage and distribution network measured by on-line, continuous disinfectant residual instruments; Effective cross-connection programs, leak detection surveillance, and pressure management practices; and An overall water safety plan approach, e.g. WHO (2009, 2011), Australian Drinking Water Guidelines (NHMRC, 2011) providing strategies for specific water systems is the best way to prevent waterborne disease outbreaks based on fully knowing and understanding that system.
Recommendations for ‘‘myth busting’’
Abandon total coliforms as a credible health risk indicator and eliminate any health advisory based solely on coliform-positive water samples – use only as general water quality parameter post-treatment in distribution systems; Replace fecal coliforms with E.coli as a more credible indicator of the possible presence of human pathogens; Abandon the belief that enumeration methods for treated drinking water provide more credible data than P/A for public health decisions; Abandon the belief that pathogen monitoring in treated drinking water will ever provide sufficient and credible data, in isolation, to make correct health risk determinations because poor PPV on treated water is inevitable.
7
Advocate much greater on-site use of CE methods for E. coli detection to informed and trained operators who can act more quickly to identify potential health risks; Eliminate barriers to water system operators to use accepted CE methods and not require water samples be sent to a certified laboratory; Use total coliforms to monitor significant water quality changes in drinking water during the distribution/storage and assess distribution system management practices to minimize undesirable deterioration, e.g. sudden loss of disinfectant residual; Rely only on clinical and epidemiological evidence on which specific microorganisms found in treated drinking water truly pose a unreasonable health risk when detected at sufficient numbers and virulence for an infective dose; Understand that molecular methods are still under development and not ready for routine monitoring purposes until questions such as interpretation of data, method standardization, certifying authority, etc., have been resolved; Accept only the medically-accepted definition for immunocompromised individuals that does not include the generalization of the ‘‘young and elderly’’. The very young and the elderly may, based on waterborne outbreak experience, be more vulnerable to severe outcomes for some pathogens, e.g. E. coli O157:H7; and Those within health organizations must base regulations and guidelines on published science and maintain a current expertise on microbial issues related to drinking water. Some current regulations and guidelines lag far behind the most recent science and are therefore not protecting public health.
Declaration of interest The authors report no conflicts of interest. Dr. Edberg is the inventor of Colilert but has no financial interest in this product. The authors alone are responsible for the content and writing of this article.
References Albrecht JA, Hamouz FL, Summer SS, Melch V. (1995). Microbial evaluation of vegetable ingredients in salad bars. J Food Protect 58: 683–5. Allen MJ, Taylor RH, Geldreich EE. (1980). The occurrence of microorganisms in water main encrustations. J AWWA 72:614–25. Allen MJ, Clancy JL, Rice EW. (2000). The plain, hard truth about pathogen monitoring. J AWWA 92:64–76. Allen MJ, Edberg SC, Reasoner DJ. (2003). Heterotrophic plate count bacteria – what is their significance in drinking water? Int J Food Microbiol 92:265–74. Allen MJ, Payment P, Clancy JL. (2010). Rapid microbial methods can improve public health protection. J AWWA 102:44–51. APHA, AWWA, and WEF. Standard methods for the examination of water and wastewater, 22nd ed. Washington, DC; 2012. Beuchat LR, Brackett RE. (1990). Inhibitory effects of raw carrots on Listeria. Appl Environ Microbiol 56:1734–42. Borchardt MA, Spencer SK, Kieke BA, et al. (2012). Virus in nondisinfected drinking water from municipal wells and community incidence of acute gastrointestinal illness. Environ Health Persp 12: 1273–9. Brenner KP, Rankin CC. (1990). New screening test to determine the acceptability of 0.45-mm membrane filters for analysis of water. Appl Environ Microbiol 56:54–64.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
8
M. J. Allen et al.
Burlingame GA, McElhaney J, Bennett M, Pipes WO. (1984). Bacterial interference with coliform colony sheen production on membrane filters. Appl Environ Microbiol 47:56–60. Callister SM, Agge WA. (1987). Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce. Appl Environ Microbiol 53:249–53. Christian RR, Pipes W. (1983). Frequency of distribution of coliforms in water distribution systems. Appl Environ Microbiol 45:603–9. Dufour AP. (1977). Escherichia coli: the fecal coliform. In: Hoadley AW, Dukta BJ, eds. American Society for Testing and Materials, Special Technical Pub. 635. Philadelphia, PA, 48–58. Duncan IBR. (1988). Waterborne Klebsiella and human disease. Toxic Assess 3:581–98. Duncan HE, Edberg SC. (1995). Host-microbe interaction in the gastrointestinal tract. Crit Rev Microbiol 21:85–100. Edberg SC, Patterson JE, Smith DB. (1994a). Differentiation of distribution systems, source water, and clinical coliforms by DNA analysis. J Clin Microbiol 321:139–42. Edberg SC, Allen MJ, Smith DB. (1994b). Rapid, specific, defined substrate for the simultaneous detection of total coliforms and Escherichia coli. Toxic Assess 3:565–80. Edberg SC, Rice EW, Karlin RJ, Allen MJ. (2000). Escherichia coli: the best biological drinking water indicator for public health protection. J Appl Microbiol 88:106S–16S. Edberg SC, Browne FA, Allen MJ. (2007). Issues for microbial regulation: Aeromonas as a model. Crit Rev Microbiol 33:89–100. Elmund GK, Allen MJ, Rice EW. (1999). Comparison of Escherichia coli, total colifroms, and fecal coliform popualtions as indicators of wastewater treatment efficiency. Water Environ Res 71:332–9. Frankland P, Frankland P. (1894). Micro-organisms in water. London & New York: Longmans, Green, and Co. Garg N, Churey JJ, Splittstoeser DF. (1990). Effect of processing conditions on the microflora of fresh-cut vegetables. J Food Protect 56:701–3. Hardalo C, Edberg SC. (1997). Pseudomonas aeruginosa: assessment of risk from drinking water. Crit Rev Microbiol 2:47–75. Hill VR, Narayanan J, Vinje´ J, Cromeans T. (2010). Sample preparation methods for molecular techniques for drinking water. Report 3108. Water Research Foundation, Denver. Hrudey SE, Leiss W. (2003). Risk management and precaution: insights on the cautious use of evidence. Environ Health Persp 111:1577–81. Hrudey SE, Rizak S. (2004). Rapid analytical techniques for drinking water security investigations. J AWWA 96:110–13. Hrudey SE, Allen MJ, Brecher RW, et al. (2008). Turbidity and microbial risk in drinking water – ministerial technical advisory committee for the Minister of Health pursuant to Section 5 of the Drinking Water Act (S.B.C.2001), Victoria, British Columbia. Iowa State College. (1921). Iowa State College Engineering Experiment Station Bulletin, Vol. 16, p. 79. Jacobs NJ, Ziegler WL, Stukel TA, et al. (1986). Comparison of membrane filter, multiple fermentation tube, and presence/absence techniques for detecting total coliforms in small community water systems. Appl Environ Microbiol 53:1571–3. Johnson CH, Rice EW, Reasoner DJ. (1997). Inactivation of Helicobacter pylori by chlorination. Appl Environ Microbiol 63: 4969–70. Kelley J, Graham K, Paterson R, et al. (2003). Identification and control of fungi in distribution systems. Water Research Foundation, Denver, CO; Report 90925, pp. 174. Monge R, Chinchilla M. (1996). Presence of Cryptosporidium oocysts in fresh vegetables. J Food Protect 59:202–3. Mulder RR, Yu VL, Wu AH. (1986). Mode of transmission of Legionella pneumophila – a critical review. Arch Intern Med 146:1607–12. Nagy LA, Olson BH. (1982). The occurrence of filamentous fungi in drinking water distribution systems. Can J Microbiol 28:667–71. Nagy LA, Olson BH. (1985). Occurrence and significance of bacteria, fungi and yeasts associated with distribution pipe surfaces. Proceedings of the American Water Works Association Water Quality Technology Conference, Denver, CO. Nagy LA, Olson BH. (1986). A comparison of media for the enumeration of filamentous fungi from aqueduct biofilm. Zentralbl Bakteriol Hyg 182:478–84. National Health & Medical Research Council (NHMRC). (2011). Australian Drinking Water Guidelines. Canberra, Australia.
Crit Rev Microbiol, Early Online: 1–8
Available from: http://www.nhmrc.gov.au/guidelines/publications/eh5 [last accessed 18 Oct 2013]. Nocker A, Burr A, Camper AK. (2010). Synthesis document on molecular methods for the drinking water industry. Report 91255. Water Research Foundation, Denver, CO. Payment P, Sartory DP, Reasoner DJ. (2003). History and use of HPC. In: Bartram J, Cotruvo J, Exner M, et al. editors. HPC and drinking-water safety – the significance of heterotrophic plate counts for water quality and human health. WHO Emerging Issues in Water and Infectious Diseases. London: IWA Publishing, pp. 20–48. Payment P, Pintar K. (2006). Waterborne pathogens: a critical assessment of methods, results, and data analysis. J Water Sci 19:233–46. Reina LD, Fleming HP, Humphries EG. (1995). Microbiological control of cucumber hydrocooling water with chlorine dioxide. J Food Protect 58:541–6. Rice EW, Fox KR, Nash HD, et al. (1987). Comparison of media for recovery of total coliform bacteria from chemically-treated water. Appl Environ Microbiol 53:1571–3. Rice EW, Geldreich EE, Read EJ. (1989). The presence–absence coliform test for monitoring drinking water quality. Pub Health Rpts 04:54–8. Rizak,S, Hrudey SE. (2006). Misinterpretation of drinking water quality monitoring with implications for risk management. Environ Sci Technol 40:5244–50. Rizak S, Hrudey SE. (2007). Evidence of water quality monitoring: limitations for outbreak detection. J Australian Inst Environ Health 7: 11–21. Rochelle PA, Clancy JL. (2006). The evolution of microbiology in the drinking water industry. J AWWA 98:163–91. UK Department for Environment, Food, and Rural Affairs. (2011). Report WD0906; Review of fungi in drinking water. pp. 174. UK Water Industry Research Microbiology Database. (2008). https:// www.ukwir.org/microsheets/terms.asp United States Environmental Protection Agency (US EPA). (1989). National primary drinking water regulations: total coliforms (including fecal coliforms and E. coli); Final Rule. Federal Register, 54:27544–68. US EPA. (2012a). National Primary Drinking Water Regulations: Revisions to the Total Coliform Rule; Final Rule. Federal Register, 78:30:10270–365. http://water.epa.gov/lawsregs/rulesregs/sdwa/tcr/ regulation.cfm. US EPA. (2012b). Recreational Water Quality Criteria. U.S. Environmental Protection Agency, Office of Water 820-F-1258. Washington, DC. Available from: http://water.epa.gov/scitech/ swguidance/standards/criteria/health/recreation/upload/recreation_ document.pdf US Federal Register. (2010). National primary drinking water regulations: revisions to the total coliform rule; Proposed Rule. 75:134:40926–1016. Vescovo MC, Orsi C, Scolari G, Torriani S. (1995). Inhibitory effects of selected lactic acid bacteria on microflora associated with ready-touse vegetables. Lett Appl Microbiol 21:121–5. Vinten-Johansen P, Brody H, Paneth N, et al. (2003). Cholera, chloroform, and the science of medicine – a life of John Snow. Oxford: Oxford University Press. Wade TJ, Sams E, Brenner KP, et al. (2010). Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study. Environ Health 9:66.doi:10.1186/1476-069x-9-66. Wadhwa SG, Khaled GH, Edberg SC. (2002). Comparative microbial character of consumed food and drinking water. Crit Rev Microbiol 28:249–79. Whiley H, Keegan A, Giglio S, Benthan R. (2012). Mycobacterium avium complex – the role of potable water in disease transmission. J Appl Microbiol 113:223–32. WHO. (2011). Water safety plans, guidelines for drinking water quality, 4th ed. Chapter 2, pp. 45–78. Geneva: World Health Organization. http://who.int/water_sanitation_publications/2011/ World Health Organization (WHO). (2009). Water safety plan manual: step-by-step risk management for drinking-water suppliers. World Health Organization. www.who.int/water_sanitation_health/publication_9789241562638/en/.
REVIEW ARTICLE
Drinking water microbial myths* Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Martin J. Allen1, Stephen C. Edberg2, Jennifer L. Clancy3, and Steve E. Hrudey4 1
Water Research Foundation (retired), Denver, CO, USA, 2School of Medicine, Yale University, New Haven, CT, USA, 3J. Clancy & Associates, St. Albans, VT, USA, and 4Environmental & Analytical Toxicology, University of Alberta, Edmonton, AB, Canada
Abstract
Keywords
Accounts of drinking water-borne disease outbreaks have always captured the interest of the public, elected and health officials, and the media. During the twentieth century, the drinking water community and public health organizations have endeavored to craft regulations and guidelines on treatment and management practices that reduce risks from drinking water, specifically human pathogens. During this period there also evolved misunderstandings as to potential health risk associated with microorganisms that may be present in drinking waters. These misunderstanding or ‘‘myths’’ have led to confusion among the many stakeholders. The purpose of this article is to provide a scientific- and clinically-based discussion of these ‘‘myths’’ and recommendations for better ensuring the microbial safety of drinking water and valid public health decisions.
Drinking water, microbial monitoring, pathogens, public health decisions, waterborne disease
Introduction The provision of a safe and sustainable drinking water supply is one of the hallmarks of a successful society. Without safe drinking water, public health is always at risk and the economic potential of the community cannot be realized. In 1849, Dr. John Snow demonstrated that cholera was attributed to drinking water contaminated by microbial pathogens by dispelling the prevailing myths of his time that cholera was caused by ‘‘odorous vapours’’ (VintenJohansen et al., 2003). Since then, scientists, engineers and public health officials have endeavored to determine which water treatment processes will remove or inactivate human pathogens in drinking water, and how best to monitor these processes and the water distributed to consumers. This article describes: the chronology of microbial monitoring practices thought to best ensure safe drinking water; key references that support or refute current monitoring approaches; discussion of the ‘‘myths’’ that have evolved and continue to be perpetuated contrary to the published science; and how these ‘‘myths’’ can hamper making correct and timely public health decisions. Drinking water microbial ‘‘myths’’ include: Routine pathogen monitoring is a credible tool to identify potential health risks;
Address for correspondence: Martin J. Allen, Water Research Foundation (retired), 6666 West Quincy Ave, Denver, CO, USA. Tel: 303-9066951. E-mail: [email protected] *The authors dedicate this paper to Dr. Donald J. Reasoner, Chief – Microbial Contaminants Branch at the USEPA, inventor of R2A HPC media for HPC bacteria, recipient of numerous awards for scientific advances in drinking water microbiology, and a fellow ‘‘myth buster’’.
History Received 24 April 2013 Revised 24 September 2013 Accepted 25 September 2013 Published online 25 November 2013
The presence of coliforms or fecal coliforms always signifies an elevated health risk; Enumeration of indicator bacteria in treated drinking water is necessary to identify potential health risk; Certain microorganisms when found in drinking water pose an increased health risk; The ‘‘very young’’ and ‘‘elderly’’ are often considered ‘‘immunocompromised’’; Molecular microbial methods will soon offer the promise of rapid detection of pathogens to make correct and timely public health decisions; Only certified laboratories are able to accurately analyze for coliforms and E. coli; and Prescribed regulatory microbial monitoring frequencies are sufficient to correctly assess health risk. Key references with in-depth data/discussion provide a broader understanding as to the origin of these myths.
Evolution of indicators to protect public health As early as the late 1800s, it was realized that monitoring for all known human pathogens (many pathogens were unknown at that time, e.g. viruses, Legionella, Cryptosporidium), was impossible and that an alternative approach for routine monitoring of the microbial safety of drinking water was necessary. Frankland & Frankland (1894) stated ‘‘The longer the bacteriological examination of water is practiced the more evident does it become that in searching for pathogenic organisms special methods must be devised and adopted according to the nature of the particular microbe of which we are in quest, and that only any possibility of such pathogenic bacteria being found in the course of ordinary plate cultivations made with natural waters, the colonies of the common
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
2
M. J. Allen et al.
water-bacteria almost invariably so predominating as to exclude all others present in small numbers’’. It was also noted the need for ‘‘ . . . larger volumes of water, as in this manner, the chance of discovery are correspondingly increased’’. And still today, pathogen monitoring of drinking water to make timely and correct public health decisions is not realistic, although some continue to believe that such monitoring may be possible. Allen et al. (2000), Payment & Pintar (2006), and Rizak & Hrudey (2006, 2007) described the major issues that make pathogen monitoring in drinking water impractical or currently ineffective for protecting public health. These include: Diversity of human pathogens, i.e. protozoa, bacteria, viruses, that may be present in drinking water, and therefore the multitude of complex standardized methods that would be necessary to successfully monitor for each type of pathogen. The extremely low numbers of pathogens in surface waters and wastewaters in comparison to the entire total microbial flora in these waters. The lack of standard methods that provide credible data, i.e. specificity, sensitivity and reproducibility, to validate the effectiveness of the in-place water treatment process and to make correct and timely public health decisions. The inability of methods to determine whether pathogens are infectious, viable, or human or non-human infectious strains. The need to examine large volumes (41.0–100 L) of water to have any reasonable chance of detecting the target pathogen. The de-sensitization of any method involved with concentrating large volumes for subsequent analysis. Very few water laboratories have resources (equipment, biohazard-rated facilities and qualified analysts) capable of performing wide-spectrum pathogen monitoring. Lack of organizations qualified and with the on-going resources to certify pathogen methods, and lack of standards. The days to weeks required to obtain a test result with sufficient accuracy to make informed public health decisions based on the monitoring data. Positive Predictive Values (PPV), the probability that a given pathogen ‘‘detection’’ is accurate for routine treated water quality monitoring for pathogens is inevitably unacceptably low. The concept of PPV (Hrudey & Leiss, 2003; Hrudey & Rizak, 2004) assesses the credibility of health risk data as it relates to the accuracy of analytical data with the method used and the probability the target contaminant (biological or nonbiological) is present frequently enough for the analytical method to provide accurate detection. All analytical methods and those especially used for detection of pathogens have significant false positive and false negative rates that compromise the credibility of the data to make correct public health decisions. Furthermore, such methods do not always determine viability, infectivity and human versus non-human strains. In the case of treated drinking water, where occurrence of pathogens should be rare, most samples tested will be truly negative, but monitoring predominately negative samples
Crit Rev Microbiol, Early Online: 1–8
with a method that has a non-zero false positive rate assures that a substantial number of false positives will occur. The rarer the occurrence of pathogens and/or the higher the false positive rate, the larger will be the number of false positives relative to the rare true positives. This inevitable reality with false positives exceeding true positives means the PPV will be much less than 50% (equal likelihood of a correct or erroneous result). For pathogen monitoring in treated drinking water, the PPV will commonly be much less than 10%, less than one chance in 10 of the result being true (Rizak & Hrudey, 2007). With all the above noted barriers to credible pathogen monitoring known, although many are dismissed even today, indicator or surrogate bacteria are used to signal recent fecal contamination and thus the possible presence of pathogens. (Edberg et al., 2000; Rochelle & Clancy, 2006). The criteria of a suitable sanitary bacterial indicator include: Always present in animal and human feces; Present in high numbers for higher probability of detection; Persistence in the environment/drinking water similar to that of pathogens; Does not multiply in the environment; and Simple, rapid, accurate, and inexpensive methods are available. Today the most widely used indicators for drinking water safety and quality include: Total Coliforms Fecal Coliforms – the correct term is ‘‘thermotolerant coliforms’’ Escherichia coli Heterotrophic Plate Count (HPC) bacteria Biological indicators other than E. coli including Enterococcus (or fecal Streptococcus), Clostridium perfringens spores, somatic coliphages and male-specific coliphages have been suggested, but may not be suitable or practical for routine monitoring of drinking water (Edberg et al., 2000), i.e. complex methods, methods not standardized, data interpretation (does the mere presence of these microorganism constitute a true health threat from consumption of drinking water?). The functional definition of total coliforms in Standard Methods for the Examination of Water and Wastewater, 22nd edition (APHA, AWWA, WEF, 2012) is the ability to metabolize lactose to produce acid and gas, or possession of the enzyme b-galactosidase (only E. coli possesses b-glucuronidase). The coliform genera and their sources are: Escherichia – human and animal feces (predominant facultative anaerobic bacterium in the colon of all warm-blooded mammals; 108–109/g in feces); Enterobacter – predominately environment, little in feces; Klebsiella – predominately environment, little in feces; Citrobacter – environment; and Serratia – environment. Analysis for total coliforms in drinking water can be performed per Standard Methods using: (1) Multiple Tube Fermentation (MTF) method; (2) Membrane Filtration (MF) method, and (3) Chromogenic/Enzymatic (CE) method. Of these methods, the MTF method is the oldest and, for drinking water, has been largely replaced by either the MF or CE
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
DOI: 10.3109/1040841X.2013.849655
Drinking water microbial myths
3
Figure 1. Scanning electron micrographs (SEM) showing bacteria colonizing a water main tubercles – New Haven, CT (Allen et al., 1980).
methods since the MTF method is labor-intense, timeconsuming and does not provide better data (see section 9221C, Standard Methods, Table 9221:II, Table 9221:III, Table 9221:IV – 95% Confidence Limits). Also the adoption of the Presence/Absence concept by many national health organizations eliminates the need for enumeration data (see later discussion). For drinking water analysis, it is common practice to use the membrane filter technique (Standard Methods-9222B) using LES Endo agar. Further in 9222B-4f, coliform verification is recommended since ‘‘typical sheen colonies may be produced occasionally by noncoliform organisms’’. Also ‘‘Atypical colonies (dark red or nucleated colonies without sheen) occasionally may be coliforms. Preferably, verify all typical atypical colony types, but at a minimum, verify all typical and five atypical colonies per membrane’’. Further, ‘‘For drinking water, verify all colonies on Endo media by swabbing the entire membrane or picking at least five typical colonies and five atypical colonies from a given filter culture. Verification methods are described in 9222B-4f require additional time (up to 48 h). The requirement to verify ‘‘typical and atypical’’ coliforms colonies from drinking water samples may not be routinely practiced with health advisories issued without verified microbial data. It is ill-advised and could be considered irresponsible to issue health advisories without conclusive microbial evidence of elevated risk or other risk factors, e.g. chlorination failure, water quality indicators, unexplained disease. The MF method is often used because it has been assumed that enumeration, i.e. number of colonies on a membrane provides data to ensure correct public health decisions. However, the time required (48 hr) to complete verification per Standard Methods when using the MF method greatly compromises timely and correct public health decisions. In contrast, the enzymatic (chromogenic) methods for coliforms and E. coli, described later and included in Standard Methods, provide verified data in 24 hr or less.
Heterotrophic bacteria and the coliform genera Enterobacter, Klebsiella and Serratia are known to grow post-treatment in the water distribution networks (Allen et al., 1980; Edberg et al., 1994a) when there are sufficient nutrients, e.g. 410 mg/L of assimilable organic carbon (AOC) for unchlorinated systems and 4100 mg/L AOC for chlorinedisinfected systems; warmer water temperatures (415 C) often found during summer months; long residence time of the water within the distribution system/storage reservoirs (days to weeks); and low disinfectant residuals especially at the far reaches of the distribution system (Figure 1a and b). Thus, total coliforms cannot be relied upon as an indicator of fecal contamination (elevated health risk) and the mere presence of total coliforms should not be used to trigger a health advisory. The fecal coliform method with higher temperature incubation of cultures was thought to favor the growth of only fecal-associated E. coli and inhibit growth of the other four coliform genera. This assumption was incorrect since up to 15% of Klebsiella (non-fecal origin) can grow at 44.5 C and up to 10% of E. coli are unable to grow at 44.5 C, thus a potential of a 25% error (false positive or false negative) (Edberg et al., 1994b). It was found that fecal coliform or more appropriately ‘‘thermotolerant coliform’’ was a poor surrogate for E. coli and has subsequently been removed from drinking water regulations. The same sciencebased rationale is appropriate for elimination of the use of the fecal coliform method for determining waste water quality. As shown above, only E. coli is extensively found in fecal matter and thus best supports its logical preference as an indicator of the possible presence of pathogens in drinking water. Tables 1 and 2 support the relevance of E.coli as the best indicator of fecal origin. A much earlier study (Iowa State College, 1921) reported the percentages of fecal types of coliform bacteria as 94.1% in human feces and 92.6% in animal feces. In both studies (Iowa State College, 1921 and Dufour, 1977) spanning 56 years, the percentage of the E. coli populations among coliform bacteria in fecal matter is consistently greater than
4
M. J. Allen et al.
Crit Rev Microbiol, Early Online: 1–8
Table 1. Relative number of fecal and nonfecal types of coliform bacteria in various substances.
Source observed Human feces Animal feces Water Milk Grain Soil
Table 3. Bacterial populations in uncooked vegetables log/gram (total coliforms, fecal coliforms, HPC bacteria). Vegetable
Number of strains
Percentage Aerobacter aerogenes*
Percentage Escherichia coli
2534 1832 2137 1382 288 853
5.9 7.4 35.2 43.1 81.7 88.1
94.1 92.6 64.8 56.9 18.3 11.9
Alfalfa sprouts Broccoli Cabbage Carrots Cauliflower
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Celery
*Renamed Enterobacter aerogenes Source: Iowa State College (1921).
Cucumber Lettuce
Table 2. Percentages of coliform genera in human and animal feces (Dufour, 1977). Mixed Salad
Animal (Number Examined)
E.coli (%)
Klebsiella spp. (%)
Enterobacter/ Citrobacter (%)
Chicken (11) Cow (15) Sheep (10) Goat (8) Pig (15) Dog (7) Cat (7) Horse (26) Human (26) Average
90 99.9 97 92 83.5 91 100 100 96.8 94.5
1 – – 8 6.8 – – – 1.5
9 0.1 3 – 9.7 – – – 1.7
Radish Spinach Tomato
# Tested 2 3 1 36 8 12 1 9 36 2 4 4 8 4 7 8 36 1 3 12 8 16 8 36
TC
ND ND 44.0 0.6–43.0 ND ND 6.3 ND ND 2.4 ND ND ND ND ND ND 4.9 ND ND ND ND ND 5.2 ND ND 4.2 ND ND ND ND ND 5.4 5.4 ND ND ND 6.1 5.5 6.2 ND ND 5.6 ND ND ND 2.6 5.2 ND
Irrelevance of enumerating microbes for drinking water safety The membrane filtration (MF) method has been the preferred method for decades since it was thought to accurately enumerate coliforms, fecal coliforms and E. coli. This assumption is not supported from statistical data although many microbiologists, engineers, public health officials, etc. remain unconvinced, i.e. enumeration is needed to make
HPC
Reference
8.2 8.8 4.5 6.6 ND 5.8 7.3 4.8 5.2 6.0 4.5 5.9 ND 5.2 6.2 ND 5.7 5.9 7.1 8.3 ND 5.9 ND 5.5
Callister & Agger (1987) Reina et al. (1995) Callister & Agger (1987) Albrecht et al. (1995) Monge & Chinchilla (1996) Garg et al. (1990) Beuchat & Brackett (1990) Garg et al. (1990) Albrecht et al. (1995) Callister & Agger (1987) Garg et al. (1990) Reina et al. (1995) Monge & Chinchilla (1996) Garg et al. (1990) Callister & Agger (1987) Monge & Chinchilla (1996) Albrecht et al. (1995) Garg et al. (1990) Vescovo et al. (1995) Beuchat & Brackett (1990) Monge & Chinchilla (1996) Garg et al. (1990) Monge & Chincilla (1996) Albrecht et al. (1995)
ND, not determined. *Provided by Dr. Eugene W. Rice, USEPA.
Table 4. 95% Confidence limits (CL) based on colony-forming units (CFU) for membrane filtration (MF) results using 100 mL samples (abridged from Standard Methods – Table 9222 III). Number of CFUs counted
93%. In particular, the 1977 data (Dufour, 1977) shows the percentage of Klebsiella spp. and Enterobacter/Citrobacter spp. among coliform bacteria in fecal matter is much lower than E. coli, making these species poor indicators of fecal contamination. Despite the overwhelming evidence that E. coli is the best indicator of potential health risk, i.e. possible presence of pathogens, total coliform monitoring for drinking water continues to be considered as a suitable indicator of possible health risks by some health agencies and public health officials. While historically the safety of drinking water has focused on the presence of total coliforms and fecal coliforms and not total bacteria, foods, in contrast, routinely contain high populations of bacteria. All foods contain very high numbers and a wide spectrum of microorganisms that rarely pose a health risk (Wadhwa et al., 2002). Coliforms, fecal coliforms and heterotrophic plate count (HPC organisms) are not unique to drinking water but are the normal flora found in the environment including vegetables that are consumed uncooked without adverse health effects. Table 3 provides such information.
FC
4 10* 20
Lower CL
Upper CL
1.0 4.7 12.2
10.2 18.4 30.8
*Ten colony forming units (CFUs) have a confidence range of 4.7–18.4 CFUs.
correct public health decisions; Table 4 shows the confidence limits of the MF method. Studies (Brenner & Rankin, 1990; Burlingame et al., 1984; Elmund et al., 1999; Jacobs et al., 1986; Rice et al., 1987; Rice et al., 1989) showed membrane filtration methods often recovered fewer target organisms than liquid methods for a variety of reasons including flawed membranes (pores too small/large, inhibitory residues); other high HPC bacteria (4500 CFU/mL) in a sample can desensitize lactose-based method (false negatives); and liquid-based methods recovery, i.e. enzymatic (chromogenic) is generally higher than MF methods (Edberg, 1994b). With the known inherent statistical variability of microbes in water samples shown in Figure 2 (Christian & Pipes, 1983), the United States Environmental Protection Agency (USEPA, 1989) adopted the presence/absence (P/A) concept for compliance monitoring as have other equivalent health organizations. In Figure 2, in a random dispersion of thirtysix 100-mL samples of water containing coliforms, the probability of one 100-mL sample having bacteria was: 37%, one bacterium; 37%, no bacteria; 18%, two bacteria and 7%, 42 bacteria. As bacteria are particulates, they are not uniformly distributed (randomness), and the enumeration of bacteria cannot be related to level of health risk. As such the
DOI: 10.3109/1040841X.2013.849655
Drinking water microbial myths
5
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Figure 2. Variability in bacterial counts detected in 100 mL grab samples from a well-mixed drinking water bulk sample (Christian & Pipes, 1983).
concept of P/A has been adopted as the science-based approach for assessing risk, thus questioning the practice of enumeration of indicator bacteria by the MF technique with its flaws noted above to be able to make a more accurate determination of potential health risk.
Public health relevance of coliforms and non-coliforms in drinking water Based on the literature for assessing the safety of drinking water, the following conclusions are made: Only the presence of E. coli in drinking water, as an indicator of fecal contamination, has potential public health significance (Edberg et al, 2000); USEPA uses E. coli only to assess potential health risk in the Revised Total Coliform Rule in 2016 (Federal Register, 78 (30), Feb 13, 2013). Total coliforms will be used as a nonhealth based water quality indicator for water treatment and distribution systems. HPC bacteria in drinking water have no public health significance, but can be used to monitor significant water quality changes as drinking water is distributed to consumers. (Allen et al., 2003). Bacteria that may be found in treated drinking water that have no public health significance from ingestion include: Klebsiella (Duncan, 1988); Aeromonas (Edberg et al., 2007); Pseudomonas aeruginosa (Hardalo & Edberg, 1997); Helicobacter (Johnson et al., 1997); HPC bacteria (Allen et al., 2003; Payment et al., 2003). The respiratory illness, Legionnaires’ disease, has often been associated with drinking water as the source of the organism and the bacterium can be isolated from even the most aggressively treated drinking water. It is important to understand the epidemiologicallyproven mode of infection is air-borne, i.e. shower aerosols, not consumption of drinking water (Mulder et al., 1986). Although Legionella can be found occasionally in treated drinking water, it is the improper distribution and storage within buildings and lack of World Health Organization (WHO)-recommended water quality management plans within buildings (hospitals, nursing homes, high rises) that promote growth of Legionella that can result in a higher risk of disease. The similar situation applies to Mycobacterium avium complex – MAC (Whiley et al., 2012) that may be present in drinking water, posing little if any health risk
from ingestion, but may pose greater health risks related to warm-water distribution systems, showers, swimming pools, hot tubs, spas, etc. Fungi are another group of microorganisms present in treated drinking water. They occur naturally in the environment, all surface waters, foods, air and some in treated drinking water (Kelley et al., 2003; Nagy & Olson, 1982, 1985, 1986; UK Department for Environment, Food & Rural Affairs, 2011; UK Water Industry Research Microbiology Database (UKWIR), 2008). These organisms are usually present in low numbers in drinking water distribution systems and are associated with taste and odor complaints, but not a direct health concern. The UKWIR database states ‘‘Effective water treatment and maintaining the integrity of the distribution systems will prevent most micro-fungi and yeast from entering water supplies’’. Further, ‘‘Maintaining an effective disinfectant residual will help to prevent the growth of fungi’’. At this time there is no clinical evidence to suggest that fungi present in drinking water poses a health risk. Immunocompromised populations It is often broadly and incorrectly stated that immunocompromised individuals include the very young/infants and the elderly. The general term immunocompromised is meaningless unless the specific immune defect is known and measureable. Immunocompromised individuals are people with significantly measureable decreases in their immune function and include patients on active anti-cancer drugs, HIV/AIDS or other chemotherapies, plus the undiagnosed, e.g. HIV/AIDS. A hematology profile showing abnormal values for gamma globulins, white blood cells, red blood cells, liver function, etc., identifies an immunocompromised individual. Avoiding consumption of treated drinking water is not necessarily recommended given the natural microflora in foods and the robustness of the host defenses of the gastrointestinal tract (lysozyme, acid pH, proteolytic enzymes, bile, etc.) (Duncan & Edberg, 1995). Individuals who are truly immunocompromised per a medical tenet are aware of their condition and should be advised by their doctor to take appropriate precautions. While the young and the elderly may be sensitive populations showing a higher prevalence of more severe outcomes in some outbreak scenarios, it is not possible to define susceptibility of the
6
M. J. Allen et al.
young or elderly accurately by age. Rather susceptibility must be defined by a physiological state – diabetes, obesity, hypertension, etc. Thus, regulations to minimize health risk from drinking water need to be based on the medically-based science as to specific populations at risk, not generalized, non-definable populations.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
Relevance and status of molecular techniques for assessing presence of human pathogens in drinking water For decades the use of molecular methods to rapidly identify human pathogens in drinking water has been thought to be a promising public health approach. Since the 1990s, the Water Research Foundation (www.WaterRF.org) funded 47 projects that involve molecular techniques with a value over $7.8 million (personal communication, John Albert, Water Research Foundation). These projects have touched upon virus, protozoa and bacterial detection in drinking water. Many other research organizations worldwide have also funded projects on developing molecular methods. There is progress in using molecular methods in support of Quantitative Microbial Risk Assessments (determining the adequacy of in-place water treatment processes to remove/ inactivate pathogens from drinking water) and use of quantitative polymerase chain reaction (qPCR) methods as an epidemiological tool (presence of enterococci, Bacteroides, enteric viruses) for recreational waters (Borchardt et al., 2012; Wade et al., 2010; US EPA, 2012b). With regard to drinking water, there are several challenges that need to be addressed and understood when considering the use of molecular methods to assess potential health risks that may be associated with viable pathogens in drinking water. Some critical questions include: Of the array of pathogens which one(s) should be selected to best determine risk? What is an appropriate protocol for analyzing for targeted organisms? Does that presence of microbial nucleic matter (fragments of DNA/RNA) in drinking water pose a health risk, i.e. capable of causing disease? What ‘‘positive’’ result or signal would imply an increased health risk? How would molecular methods be certified/approved based on standardized criteria to ensure sensitivity, specificity, reproducibility and by which certifying organizations? What criteria (appropriate resources, technical staff and methods training/experience) are essential to routinely use molecular methods with confidence? What are the sample preparation and storage issues, e.g. loss of sensitivity during concentration procedures that need to be addressed to ensure credible data? Is the PPV adequate to make credible public health decisions? Two published studies on molecular methods (Hill et al., 2010; Nocker et al., 2010) reported: Water utility laboratory managers view molecular methods favorably with the major benefit being time to detection;
Crit Rev Microbiol, Early Online: 1–8
Despite the favorable impression, many managers stated that their laboratories do not currently have the resources or expertise to use these methods; Researchers and utilities surveyed by the United States Centers for Disease Control and Prevention using molecular methods for drinking water monitoring see routine pathogen screening possible within the next 10 years provided barriers are overcome, e.g. method standardization, sample preparation issues, such as loss of sensitivity during concentration procedures (Hill et al., 2010).
Rapid microbial methods can improve public health protection Beginning in the late 1980s, a new type of microbial analysis drawing from clinical technologies was developed based on CE that simultaneously measure the P/A of both total coliforms and E. coli. The use of CE methods along with sanitary surveys can greatly enhance public health protection, especially for smaller remote communities found in every country; for example 93% in the US and 75% of Canadian public water systems serve 510 000 people (Allen et al., 2000). Several CE methods are approved for detection of total coliforms and E. coli including: ColilertÕ , Colilert-18Õ , ReadycultÕ , E*Colite TestÕ , ColitagÕ , and ColisureÕ . Despite the enormous promise of enhanced public health protection for small to medium-sized water utilities by using CE methods, there are many indefensible and erroneous barriers that prevent their greater use (Allen et al., 2010). These unwarranted barriers to wider adoption include beliefs that: (1) Utility operators do not have ability or training; (2) Operators may ‘‘cheat’’ to ensure negative results; (3) Cost of equipment and media is prohibitively expensive; (4) Certified laboratories may have fewer samples to process with loss of income; (5) The laboratory certification authority will be perceived as having less control; (6) Public health authority could be perceived as abrogating their oversight; and (7) Current regulations require samples be sent to certified laboratories. CE methods are successfully used by small and remote water systems in Alaska and First Nations in Quebec Province of Canada (Allen et al., 2010). The elimination of these barriers, particularly regulatory constraints, would provide a much higher level of public health protection especially for the medium to smaller water systems where access to certified laboratories is severely limited. If there is a genuine interest to protect health protection rather than maintaining the status quo, the use of CE methods on-site by trained operators should be embraced for both large and smaller water utilities.
Unrealsitic expectations for microbial monitoring While the use of E. coli to help determine potential health risk in treated drinking water is useful and probably the best that can be done at this time, in reality, microbial monitoring as now practiced or required is a very imperfect and questionable approach to credible health protection considering: The percentage of drinking water tested for indicator bacteria is 0.0000002–0.0000005% meaning 99.99998–99.99995% is not tested;
Drinking water microbial myths
DOI: 10.3109/1040841X.2013.849655
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
The lack of plausibility in concluding an elevated health risk with a ‘‘positive’’ sample given the miniscule volume of water tested; Understanding that even monitoring using the current best indicator organism (E. coli) cannot be shown to have prevented any drinking water-borne outbreaks (Hrudey et al., 2008) – results are reported after contamination has occurred – but data on such indicators are certainly important for investigating and verifying outbreaks; Under unique situations (water main breaks, crossconnection failures, earthquakes, hurricanes, tsunamis, other catastrophic events), on-site microbial monitoring to assess health risk is appropriate; and, While microbial monitoring is one tool that can be used for assessing water quality and potential health risk, there are more suitable non-microbial water quality and treatment performance parameters (chlorine residual, turbidity) and other approaches, e.g. sanitary surveys, integrity of the water distribution network, that provide a much higher degree of confidence on the safety of drinking water. A more pragmatic and defensible approach for better ensuring safe drinking water should include: Appropriate treatment processes in place as determined by source water quality; Continuous on-line monitoring of the treatment processes that ensures the processes are optimized, e.g. use of on-line turbidimeters, disinfectant residuals at the periphery of the water distribution network. Trained and certified operators with certification to perform microbial analysis; Appropriate scheduled operation and maintenance of the treatment processes; Appropriate disinfection residuals throughout the storage and distribution network measured by on-line, continuous disinfectant residual instruments; Effective cross-connection programs, leak detection surveillance, and pressure management practices; and An overall water safety plan approach, e.g. WHO (2009, 2011), Australian Drinking Water Guidelines (NHMRC, 2011) providing strategies for specific water systems is the best way to prevent waterborne disease outbreaks based on fully knowing and understanding that system.
Recommendations for ‘‘myth busting’’
Abandon total coliforms as a credible health risk indicator and eliminate any health advisory based solely on coliform-positive water samples – use only as general water quality parameter post-treatment in distribution systems; Replace fecal coliforms with E.coli as a more credible indicator of the possible presence of human pathogens; Abandon the belief that enumeration methods for treated drinking water provide more credible data than P/A for public health decisions; Abandon the belief that pathogen monitoring in treated drinking water will ever provide sufficient and credible data, in isolation, to make correct health risk determinations because poor PPV on treated water is inevitable.
7
Advocate much greater on-site use of CE methods for E. coli detection to informed and trained operators who can act more quickly to identify potential health risks; Eliminate barriers to water system operators to use accepted CE methods and not require water samples be sent to a certified laboratory; Use total coliforms to monitor significant water quality changes in drinking water during the distribution/storage and assess distribution system management practices to minimize undesirable deterioration, e.g. sudden loss of disinfectant residual; Rely only on clinical and epidemiological evidence on which specific microorganisms found in treated drinking water truly pose a unreasonable health risk when detected at sufficient numbers and virulence for an infective dose; Understand that molecular methods are still under development and not ready for routine monitoring purposes until questions such as interpretation of data, method standardization, certifying authority, etc., have been resolved; Accept only the medically-accepted definition for immunocompromised individuals that does not include the generalization of the ‘‘young and elderly’’. The very young and the elderly may, based on waterborne outbreak experience, be more vulnerable to severe outcomes for some pathogens, e.g. E. coli O157:H7; and Those within health organizations must base regulations and guidelines on published science and maintain a current expertise on microbial issues related to drinking water. Some current regulations and guidelines lag far behind the most recent science and are therefore not protecting public health.
Declaration of interest The authors report no conflicts of interest. Dr. Edberg is the inventor of Colilert but has no financial interest in this product. The authors alone are responsible for the content and writing of this article.
References Albrecht JA, Hamouz FL, Summer SS, Melch V. (1995). Microbial evaluation of vegetable ingredients in salad bars. J Food Protect 58: 683–5. Allen MJ, Taylor RH, Geldreich EE. (1980). The occurrence of microorganisms in water main encrustations. J AWWA 72:614–25. Allen MJ, Clancy JL, Rice EW. (2000). The plain, hard truth about pathogen monitoring. J AWWA 92:64–76. Allen MJ, Edberg SC, Reasoner DJ. (2003). Heterotrophic plate count bacteria – what is their significance in drinking water? Int J Food Microbiol 92:265–74. Allen MJ, Payment P, Clancy JL. (2010). Rapid microbial methods can improve public health protection. J AWWA 102:44–51. APHA, AWWA, and WEF. Standard methods for the examination of water and wastewater, 22nd ed. Washington, DC; 2012. Beuchat LR, Brackett RE. (1990). Inhibitory effects of raw carrots on Listeria. Appl Environ Microbiol 56:1734–42. Borchardt MA, Spencer SK, Kieke BA, et al. (2012). Virus in nondisinfected drinking water from municipal wells and community incidence of acute gastrointestinal illness. Environ Health Persp 12: 1273–9. Brenner KP, Rankin CC. (1990). New screening test to determine the acceptability of 0.45-mm membrane filters for analysis of water. Appl Environ Microbiol 56:54–64.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by Southern Methodist University Cul Fond on 08/27/14 For personal use only.
8
M. J. Allen et al.
Burlingame GA, McElhaney J, Bennett M, Pipes WO. (1984). Bacterial interference with coliform colony sheen production on membrane filters. Appl Environ Microbiol 47:56–60. Callister SM, Agge WA. (1987). Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce. Appl Environ Microbiol 53:249–53. Christian RR, Pipes W. (1983). Frequency of distribution of coliforms in water distribution systems. Appl Environ Microbiol 45:603–9. Dufour AP. (1977). Escherichia coli: the fecal coliform. In: Hoadley AW, Dukta BJ, eds. American Society for Testing and Materials, Special Technical Pub. 635. Philadelphia, PA, 48–58. Duncan IBR. (1988). Waterborne Klebsiella and human disease. Toxic Assess 3:581–98. Duncan HE, Edberg SC. (1995). Host-microbe interaction in the gastrointestinal tract. Crit Rev Microbiol 21:85–100. Edberg SC, Patterson JE, Smith DB. (1994a). Differentiation of distribution systems, source water, and clinical coliforms by DNA analysis. J Clin Microbiol 321:139–42. Edberg SC, Allen MJ, Smith DB. (1994b). Rapid, specific, defined substrate for the simultaneous detection of total coliforms and Escherichia coli. Toxic Assess 3:565–80. Edberg SC, Rice EW, Karlin RJ, Allen MJ. (2000). Escherichia coli: the best biological drinking water indicator for public health protection. J Appl Microbiol 88:106S–16S. Edberg SC, Browne FA, Allen MJ. (2007). Issues for microbial regulation: Aeromonas as a model. Crit Rev Microbiol 33:89–100. Elmund GK, Allen MJ, Rice EW. (1999). Comparison of Escherichia coli, total colifroms, and fecal coliform popualtions as indicators of wastewater treatment efficiency. Water Environ Res 71:332–9. Frankland P, Frankland P. (1894). Micro-organisms in water. London & New York: Longmans, Green, and Co. Garg N, Churey JJ, Splittstoeser DF. (1990). Effect of processing conditions on the microflora of fresh-cut vegetables. J Food Protect 56:701–3. Hardalo C, Edberg SC. (1997). Pseudomonas aeruginosa: assessment of risk from drinking water. Crit Rev Microbiol 2:47–75. Hill VR, Narayanan J, Vinje´ J, Cromeans T. (2010). Sample preparation methods for molecular techniques for drinking water. Report 3108. Water Research Foundation, Denver. Hrudey SE, Leiss W. (2003). Risk management and precaution: insights on the cautious use of evidence. Environ Health Persp 111:1577–81. Hrudey SE, Rizak S. (2004). Rapid analytical techniques for drinking water security investigations. J AWWA 96:110–13. Hrudey SE, Allen MJ, Brecher RW, et al. (2008). Turbidity and microbial risk in drinking water – ministerial technical advisory committee for the Minister of Health pursuant to Section 5 of the Drinking Water Act (S.B.C.2001), Victoria, British Columbia. Iowa State College. (1921). Iowa State College Engineering Experiment Station Bulletin, Vol. 16, p. 79. Jacobs NJ, Ziegler WL, Stukel TA, et al. (1986). Comparison of membrane filter, multiple fermentation tube, and presence/absence techniques for detecting total coliforms in small community water systems. Appl Environ Microbiol 53:1571–3. Johnson CH, Rice EW, Reasoner DJ. (1997). Inactivation of Helicobacter pylori by chlorination. Appl Environ Microbiol 63: 4969–70. Kelley J, Graham K, Paterson R, et al. (2003). Identification and control of fungi in distribution systems. Water Research Foundation, Denver, CO; Report 90925, pp. 174. Monge R, Chinchilla M. (1996). Presence of Cryptosporidium oocysts in fresh vegetables. J Food Protect 59:202–3. Mulder RR, Yu VL, Wu AH. (1986). Mode of transmission of Legionella pneumophila – a critical review. Arch Intern Med 146:1607–12. Nagy LA, Olson BH. (1982). The occurrence of filamentous fungi in drinking water distribution systems. Can J Microbiol 28:667–71. Nagy LA, Olson BH. (1985). Occurrence and significance of bacteria, fungi and yeasts associated with distribution pipe surfaces. Proceedings of the American Water Works Association Water Quality Technology Conference, Denver, CO. Nagy LA, Olson BH. (1986). A comparison of media for the enumeration of filamentous fungi from aqueduct biofilm. Zentralbl Bakteriol Hyg 182:478–84. National Health & Medical Research Council (NHMRC). (2011). Australian Drinking Water Guidelines. Canberra, Australia.
Crit Rev Microbiol, Early Online: 1–8
Available from: http://www.nhmrc.gov.au/guidelines/publications/eh5 [last accessed 18 Oct 2013]. Nocker A, Burr A, Camper AK. (2010). Synthesis document on molecular methods for the drinking water industry. Report 91255. Water Research Foundation, Denver, CO. Payment P, Sartory DP, Reasoner DJ. (2003). History and use of HPC. In: Bartram J, Cotruvo J, Exner M, et al. editors. HPC and drinking-water safety – the significance of heterotrophic plate counts for water quality and human health. WHO Emerging Issues in Water and Infectious Diseases. London: IWA Publishing, pp. 20–48. Payment P, Pintar K. (2006). Waterborne pathogens: a critical assessment of methods, results, and data analysis. J Water Sci 19:233–46. Reina LD, Fleming HP, Humphries EG. (1995). Microbiological control of cucumber hydrocooling water with chlorine dioxide. J Food Protect 58:541–6. Rice EW, Fox KR, Nash HD, et al. (1987). Comparison of media for recovery of total coliform bacteria from chemically-treated water. Appl Environ Microbiol 53:1571–3. Rice EW, Geldreich EE, Read EJ. (1989). The presence–absence coliform test for monitoring drinking water quality. Pub Health Rpts 04:54–8. Rizak,S, Hrudey SE. (2006). Misinterpretation of drinking water quality monitoring with implications for risk management. Environ Sci Technol 40:5244–50. Rizak S, Hrudey SE. (2007). Evidence of water quality monitoring: limitations for outbreak detection. J Australian Inst Environ Health 7: 11–21. Rochelle PA, Clancy JL. (2006). The evolution of microbiology in the drinking water industry. J AWWA 98:163–91. UK Department for Environment, Food, and Rural Affairs. (2011). Report WD0906; Review of fungi in drinking water. pp. 174. UK Water Industry Research Microbiology Database. (2008). https:// www.ukwir.org/microsheets/terms.asp United States Environmental Protection Agency (US EPA). (1989). National primary drinking water regulations: total coliforms (including fecal coliforms and E. coli); Final Rule. Federal Register, 54:27544–68. US EPA. (2012a). National Primary Drinking Water Regulations: Revisions to the Total Coliform Rule; Final Rule. Federal Register, 78:30:10270–365. http://water.epa.gov/lawsregs/rulesregs/sdwa/tcr/ regulation.cfm. US EPA. (2012b). Recreational Water Quality Criteria. U.S. Environmental Protection Agency, Office of Water 820-F-1258. Washington, DC. Available from: http://water.epa.gov/scitech/ swguidance/standards/criteria/health/recreation/upload/recreation_ document.pdf US Federal Register. (2010). National primary drinking water regulations: revisions to the total coliform rule; Proposed Rule. 75:134:40926–1016. Vescovo MC, Orsi C, Scolari G, Torriani S. (1995). Inhibitory effects of selected lactic acid bacteria on microflora associated with ready-touse vegetables. Lett Appl Microbiol 21:121–5. Vinten-Johansen P, Brody H, Paneth N, et al. (2003). Cholera, chloroform, and the science of medicine – a life of John Snow. Oxford: Oxford University Press. Wade TJ, Sams E, Brenner KP, et al. (2010). Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study. Environ Health 9:66.doi:10.1186/1476-069x-9-66. Wadhwa SG, Khaled GH, Edberg SC. (2002). Comparative microbial character of consumed food and drinking water. Crit Rev Microbiol 28:249–79. Whiley H, Keegan A, Giglio S, Benthan R. (2012). Mycobacterium avium complex – the role of potable water in disease transmission. J Appl Microbiol 113:223–32. WHO. (2011). Water safety plans, guidelines for drinking water quality, 4th ed. Chapter 2, pp. 45–78. Geneva: World Health Organization. http://who.int/water_sanitation_publications/2011/ World Health Organization (WHO). (2009). Water safety plan manual: step-by-step risk management for drinking-water suppliers. World Health Organization. www.who.int/water_sanitation_health/publication_9789241562638/en/.
