Page 5: Guidance on Waterborne Bacterial Pathogens

Part B. Supporting information

B.2 Waterborne non-faecal pathogens

Although E. coli is the best available indicator of recent faecal contamination, there are waterborne illnesses that result from pathogens not transmitted by the faecal-oral route. These pathogens are usually bacteria naturally found in source waters. Those that clearly have a public health impact include Legionella, Mycobacterium avium complex, Aeromonas and Helicobacter pylori. The detection of faecal indicators does not provide any information on the potential presence of non-faecal pathogens. No indicators are currently known for such pathogens.

B.2.1 Legionella

Legionellae are recognized human pathogens that can cause two different types of illness: Legionnaires' disease, which is a serious respiratory illness involving pneumonia, and Pontiac fever, which is a milder flu-like illness without pneumonia. Legionellae are free-living aquatic bacteria that occur widely in water environments. The presence of Legionella is more of a concern for water systems beyond municipal water treatment and distribution systems, such as cooling towers and hospital and residential plumbing systems. Legionella species exhibit a number of survival properties that make them relatively resistant to the effects of chlorination and elevated water temperatures. The organisms are also capable of colonizing drinking water distribution system biofilms (Lau and Ashbolt, 2009).

The bacteria themselves are weakly Gram-negative, small, motile rods that have precise nutritional requirements and as a result do not grow well on culture media. At least 50 different Legionella species have been identified, and approximately half of these species have been associated with disease. Legionella pneumophila (serogroup 1) is the agent responsible for most cases of illness in humans. Other than L. pneumophila, species causing far fewer infections but still considered to be clinically relevant include L. micdadei, L. bozemanii, L. longbeachae and L. dumoffi (Reingold et al., 1984; Doyle and Heuzenroeder, 2002; Roig et al., 2003).

B.2.1.1 Sources and exposure

Legionella species are naturally present in a wide range of freshwater environments, including surface water (Fliermans et al., 1981; Palmer et al., 1993) and groundwater (Brooks et al., 2004; Costa et al., 2005). The bacteria are not considered to be enteric pathogens and are not transmitted via the faecal-oral route. However, Legionella can occasionally be detected in human faecal samples, as diarrhoea is a symptom of illness in a small percentage of cases (Rowbotham, 1998). Similarly, animals are not reservoirs for Legionella (U.S. EPA, 1999a).

Legionella can be isolated from human-made systems (e.g., cooling towers, hot water tanks, showerheads, aerators) and are most frequently associated with biofilms (Lau and Ashbolt, 2009). In general, the amount of legionellae in source waters is low compared with the concentrations that can be reached in human-made systems (Mathys et al., 2008). In an investigation of biofilm formation and Legionella colonization on various plumbing materials (Rogers et al., 1994), the detection of Legionella was greater in biofilm than in free water but varied in time and with different plumbing materials. Biofilms are important for the survival of the fastidious legionellae: they provide protection to Legionella, which are also able to utilize nutrients supplied by other organisms in this nutrient-rich environment (Borella et al., 2005; Temmerman et al., 2006; Lau and Ashbolt, 2009).

Some naturally occurring waterborne protozoa, such as Acanthamoeba, Hartmanella, Naegleria, Valkampfia and Echinamoeba, can also harbour Legionella organisms (Rowbotham, 1986; Kilvington and Price, 1990; Kramer and Ford, 1994; Fields, 1996). Legionella can infect and remain within the protozoan cyst form, where they are protected from disinfectants (Kilvington and Price, 1990; Thomas et al., 2004; Declerck et al., 2007). They are also able to multiply within these protozoa, which has been proposed as the only way that Legionella can replicate within aquatic systems (Abu Kwaik et al., 1998; Thomas et al., 2004). Thus, as well as offering protection, this association suggests a mechanism for the increase and transport of L. pneumophila in human-made systems (Declerck et al., 2009).

Temperature is an additional factor that influences Legionella colonization of water systems. Temperatures between 20°C and 50°C are hospitable for colonization, although legionellae typically grow to high concentrations only at temperatures below 42°C (Percival et al., 2004).

Plumbing systems outside of public water supply systems (e.g., in residential buildings, hotels, institutional settings) are most commonly implicated in L. pneumophila infections (Yoder et al., 2008). As Legionella is a respiratory pathogen, systems that generate aerosols, such as cooling towers, whirlpool baths and showerheads, are the more commonly implicated sources of infection. The hot water supply system is commonly pinpointed as the origin of the contamination (Hershey et al., 1997; McEvoy et al., 2000; Borella et al., 2004; Oliver et al., 2005; Burnsed et al., 2007; Yoder et al., 2008). However, the cold water supply, when held at about 25°C, which is within the range of Legionella multiplication, has also been implicated (Hoebe et al., 1998; Cowgill et al., 2005). Legionella infection can occur when people breathe in aerosolized water containing the bacteria or aspirate water containing the bacteria. The bacteria have not been found to be transmitted from person to person (U.S. EPA, 1999a).

Legionella contamination is particularly troublesome in hospitals, where susceptible individuals can be exposed to aerosols containing hazardous concentrations of L. pneumophila. Large buildings such as hotels, community centres, industrial buildings and apartment buildings are most often implicated as sources of outbreaks (Reimer et al., 2010). Studies have shown that contamination of domestic hot water systems with Legionella can occur in single-family homes (Joly, 1985; Alary and Joly, 1991; Stout et al., 1992a; Marrie et al., 1994; Dufresne et al., 2012). In a study of hot water plumbing systems in homes in the Québec area, Alary and Joly (1991) reported that Legionella was detected in 39% (69/178) of hot water tanks with electric heaters and in 0% (0/33) of tanks with oil- or gas-fired heaters. The authors further observed that in a proportion of those homes whose hot water tanks tested positive for Legionella, the organism could also be detected at distal locations, such as faucets (12%) and showerheads (15%). The position of the heat source in the design of electrically heated hot water tanks observed at the time of the study was cited as the reason for the difference in contamination between the two water heater types. In the electric tanks, the heating elements were located above the bottom of the heater, which could allow bottom sediments and water below the heating element to remain at the lower temperatures (< 50-60°C) permissible for Legionella growth (Alary and Joly, 1991). Although the presence of the bacteria in the home can increase the risk of infection in susceptible individuals, it does not necessarily mean that occupants will develop the illness. In addition, although outbreaks generally do not occur in residential settings, individual cases have been identified as originating from residential plumbing (Falkinham et al., 2008).

Similarly, evidence has been provided that sporadic cases of Legionnaires' disease can plausibly be acquired from aerosols in residential plumbing systems (Stout et al., 1992b; Straus et al., 1996; Lück et al., 2008). In a study conducted in the province of Quebec, Dufresne et al. (2012) observed that among 36 legionellosis-confirmed patients residing in homes with domestic hot water tanks, residential and clinical isolates of Legionella were microbiologically related by pulse-field gel electrophoresis in 14% (5/36) of the cases. Similar studies conducted in Pittsburgh, Pennsylvania (Stout et al., 1992b), and the state of Ohio (Straus et al., 1996) showed comparable results.

Persons thought to be at the highest risk of contracting Legionnaires' disease are those with lung conditions or compromised immune systems (e.g., persons receiving transplants or chemotherapy, persons with diabetes or kidney disease). The risk of infection is higher among persons 40-70 years of age, and the disease is seen more frequently in males than in females (Percival et al., 2004). Other risk factors include smoking and excessive use of alcohol. Legionnaires' disease is considered a very rare cause of pneumonia in children. In contrast, age, gender and smoking do not seem to be risk factors for Pontiac fever (Diederen, 2008).

The concentration of Legionella required to cause infection is not well understood (Armstrong and Haas, 2008). It has been suggested that amoebae harbouring Legionella may increase the potential for infectivity by providing a mechanism to expose humans to hundreds of Legionella cells if inhaled or aspirated in an aerosol (Rowbotham, 1986; Greub and Raoult, 2004).

B.2.1.2 Health effects

As mentioned previously, there are two distinct illnesses caused by Legionella: Legionnaires' disease and Pontiac fever. Collectively, these illnesses are referred to as legionellosis. Legionnaires' disease is a serious respiratory illness involving pneumonia. Other features include fever, cough and headache, chest and muscle pain, and a general feeling of unwellness (malaise) (Fields et al., 2002). The time from the point of infection to the onset of symptoms is about 2-10 days, and the disease period can last up to several months. One problem in diagnosing Legionnaires' disease is a lack of any specific symptom that distinguishes it from other bacterial pneumonias. Reported rates of Legionnaires' disease in Canada over the period 2000-2004 (the latest year for which data have been published) ranged from 0.13 to 0.20 cases per 100 000 population (PHAC, 2006). The mortality rate of Legionnaires' disease in the United States as of 1998 was reported at roughly 10% and 14% for community-acquired and hospital-acquired cases, respectively (Benin et al., 2002). Early diagnosis and antibiotic therapy are keys to successfully treating Legionnaires' disease.

Pontiac fever is a less serious respiratory illness that does not involve pneumonia and is more flu-like in nature. The time to the onset of symptoms is 24-48 hours (AWWA, 2006). The disease is self-limiting and typically resolves without complications in 2-5 days. No known fatalities have been reported with this illness. Pontiac fever is difficult to distinguish from other respiratory diseases because of a lack of specific clinical features. Experts have speculated that the disease may be caused by exposure to a mixture of live and dead Legionella cells and non-Legionella endotoxin (Diederen, 2008). Antibiotic treatment is typically not prescribed because of the short, self-limiting nature of the disease.

B.2.1.3 Treatment technology

Successful control of Legionella in drinking water supplies requires focused attention not only on the organisms themselves,but also on the control of free-living amoebae and biofilms that support their persistence.

Physical removal mechanisms used during drinking water treatment, such as conventional filtration (i.e., coagulation, flocculation and sedimentation), will reduce the number of Legionella present in finished water. Disinfection strategies shown to be effective in reducing the number of Legionella present include the use of chlorine, monochloramine, chlorine dioxide ozone and UV. However, it must be noted that chlorine dioxide (Health Canada, 2008) and ozone (U.S. EPA, 1999b) are generally not effective in maintaining a disinfectant residual in the distribution system and UV does not provide a disinfectant residual. In comparison with E. coli, Legionella cells have been shown to be more resistant to chlorination (Delaedt et al., 2008; Wang et al., 2010). Survival strategies exhibited by the organisms (colonization of biofilms and residence within free-living amoebae) also further protect Legionella from the action of disinfectants.

In the distribution system, currently recommended disinfectant residuals are sufficient to keep the concentration of non-biofilm-associated Legionella at levels that have not been associated with disease (Storey et al., 2004; Delaedt et al., 2008).

Various alternative disinfection methods have been examined for their potential to control Legionella colonization in municipal drinking water distribution systems. Monochloramine has been shown to be more effective than chlorine as a residual disinfectant against legionellae. Weintraub et al. (2008) observed that converting from chlorine to monochloramine for residual disinfection in a municipal distribution system resulted in a significant reduction in the number of distribution samples, point-of-use sites and water heaters positive for Legionella colonization. Pryor et al. (2004) studied the impact of changing the secondary disinfectant from chlorine to chloramine on the microbiological quality of the drinking water in a utility. Samples were taken from the source water, from the distribution system and at the point of use (i.e., showerheads). The authors found that there was a decrease in the colonization rate and the variety of Legionella species present in samples from the distribution system and showerheads when the disinfectant was changed from chlorine to choramine. However, despite the decrease in the variety of Legionella species, there was no indication of a decrease in L. pneumophila at the point of use (Pryor et al., 2004).

Kool et al. (1999) reported that hospitals supplied with water containing monochloramine for secondary disinfection were less likely to have reported outbreaks of Legionnaires' disease than those supplied with water containing free chlorine. Monochloramine is considered better able to penetrate into biofilms (LeChevallier et al., 1980), more stable and therefore able to maintain its concentration over greater distances in the distribution system (Kool et al., 1999).

It has been suggested that ozone may be a more effective disinfectant against Legionella than chlorine, but its main drawback is that it does not provide a disinfectant residual (U.S. EPA, 1999b; Kim et al., 2002; Blanc et al., 2005). Loret et al. (2005) observed that ozone at a concentration of 0.5 mg/L was effective in reducing Legionella, protozoa and biofilms in a model distribution system, but it was not as effective as chorine (2 mg/L) or chlorine dioxide (0.5 mg/L). Conversely, ozone at a concentration of 0.1-0.3 µg/mL (0.1-0.3 mg/L) was shown to be as effective as free chlorine at 0.4 mg/L in inactivating Legionella suspensions, producing a 2 log reduction of the organism within 5 minutes in laboratory experiments (Dominique et al., 1988). There are also a variety of supplemental strategies that can be used to control Legionella in the plumbing systems of large commercial, industrial and residential buildings.

Hyperchlorination has been employed as a control strategy in large buildings, including hospitals. However, studies have demonstrated that Legionella residing in biofilms or in cysts of Acanthamoeba polyphaga can survive following exposure to free chlorine at 50 mg/L (Kilvington and Price, 1990; Cooper and Hanlon, 2010). There is also the concern of an increased potential for corrosion of plumbing systems with continued high concentrations of chlorine (Kool et al., 1999).

Chlorine dioxide has demonstrated similar advantages over chlorine as a residual disinfectant for Legionella control when applied to a small distribution system such as a hospital complex. Sidari et al. (2004) observed significantly decreased Legionella concentrations at hot and cold point-of-use sites of a hospital's plumbing system upon switching to chlorine dioxide as the residual disinfectant.

In plumbing systems of hospitals and large buildings, thermal disinfection (elevating hot water to temperatures above 70°C, and flushing use points such as taps and showerheads) has been routinely employed either on its own or in conjunction with chemical disinfection. Typically this is recognized as a temporary control strategy, as Legionella recolonization can occur within weeks to months of treatment (Storey et al., 2004).

Use of copper-silver ionization systems has also received much study and has shown effectiveness in controlling Legionella in drinking water supplies (Stout et al., 1998; Kusnetsov et al., 2001; Stout and Yu, 2003; Cachafeiro et al., 2007). Stout et al. (1998) observed that copper-silver ionization (mean copper and silver concentrations of 0.29 mg/L and 0.054 mg/L, respectively, in the hot water tank) was more effective than a superheat-and-flush method in reducing the recovery of Legionella from a hospital plumbing/distribution system. In a survey of the experiences of hospital systems using copper-silver ionization, Stout and Yu (2003) reported that following installation of the disinfection systems, the percentages of hospitals reporting (1) cases of Legionnaires' disease and (2) positive Legionella samples at more than 30% of the sites measured had both been reduced to 0% from 100% and 47%, respectively, before the systems had been installed. It is important to note that use of these systems should include monitoring of copper and silver concentrations in the water, as these concentrations will increase. In addition, pretreatment may be required to address pH and hardness challenges (Bartram et al., 2007).

Additional measures used for large plumbing systems include temperature control; control of water system design and construction to prevent the accumulation of biofilms, sediments or deposits; and nutrient control strategies (Bartram et al., 2007; Bentham et al., 2007).

General recommendations regarding the control of Legionella in domestic plumbing systems involve maintaining proper water temperatures. The National Plumbing Code of Canada includes requirements of a minimum water temperature of 60°C in hot water storage tanks, to address the growth of Legionella (NRCC, 2010). Where increased hot water temperatures create an increased risk of scalding for vulnerable groups (e.g., children and the elderly), appropriate safety measures should be applied to limit the temperature to 49°C. Thermostatic or pressure-balanced mixing valves can be installed to control the water temperature at the tap to reduce the risk of scalding (Bartram et al., 2007; Bentham et al., 2007).

Control of Legionella in water systems outside of plumbing systems also requires controlling its growth in biofilms. The heating, ventilation and air conditioning industry has guidelines for reducing Legionella growth in cooling systems (ASHRAE, 2000). Hotel and lodging industry requirements for the operation and maintenance of plumbing facilities, including procedures for the proper disinfection of plumbing equipment in their facilities, are generally specified under various public health regulations and/or legislation. To obtain information on these requirements, the appropriate provincial or territorial ministry of health should be consulted.

B.2.1.4 Assessment

The increasing importance of Legionella as a cause of human infection can in part be linked to continued human development and the resulting dependence on human-made plumbing systems (Fields et al., 2002). Despite being ubiquitous in source waters, Legionella pneumophila and other Legionella species have been recovered only in low concentrations from Canadian drinking water supplies (Dutka et al., 1984; Tobin et al., 1986), and people do not get infected with Legionella by consuming drinking water. As Legionella is a respiratory pathogen, infection can occur if people breathe in contaminated aerosols. Thus, the presence of Legionella becomes a problem only when they are able to grow to high numbers in water systems such as showers, cooling towers or whirlpool baths that generate aerosols or sprays. These plumbing systems have been implicated in outbreaks, but are mainly outside of the control of municipal water treatment and distribution. For these reasons, the presence of the organism in low numbers in the distribution network is not sufficient evidence to warrant remedial action in the absence of disease cases (Dufour and Jakubowski, 1982; Tobin et al., 1986).

Owing to the existence of Legionella outside of faecal sources in nature, E. coli is not expected to be a reliable indicator of the presence of these bacteria. No suitable indicators have been identified to signal increasing concentrations of Legionella in a building's plumbing system. There is some evidence that increasing Legionella concentrations are accompanied, or preceded, by elevated heterotrophic plate count (HPC) measurements (WHO, 2002). However, the correlation between HPC and Legionella is not consistent.

Legionella have also been included on the U.S. Environmental Protection Agency's (EPA) Candidate Contaminant List as one of the priority contaminants for regulatory decision-making and information collection (U.S. EPA, 2009). Guidelines or regulations that have been developed for Legionella in Canada, the United States and other countries worldwide relate to control of the organism in water environments outside of municipal water distribution networks (e.g., piped water systems, cooling towers, health care facilities) (Cunliffe, 2007).

B.2.2 Mycobacterium avium complex

The Mycobacterium avium complex (Mac)Footnote 1 is a group of environmental mycobacteria that can cause illness in humans. The group consists of Mycobacterium avium (includes subspecies avium, sylvaticum and paratuberculosis); and Mycobacterium intracellulare (Cangelosi et al., 2004). Mac organisms are considered ubiquitous in natural waters. Transmission is primarily through contact with contaminated waters via either ingestion or inhalation (AWWA, 2006). Mac-related disease comes mainly in the form of lung infections and occurs largely in persons who have suppressed immune systems (Percival et al., 2004).

Mycobacteria themselves are motile, rod- to coccoid-shaped bacteria that have characteristically high levels of waxy lipids in their cell walls. They are Gram negative, but are more commonly considered to be "acid-fast" because of the way their cell walls respond to diagnostic staining procedures (AWWA, 2006). Mac organisms are also referred to as "non-tuberculous" or "atypical" mycobacteria. This is to distinguish them from the more well-known mycobacteria species that are responsible for tuberculosis and leprosy, which are not a concern for drinking water (Nichols et al., 2004). Other environmental mycobacteria are known that have been linked to skin infections through waterborne contact, but these are also of lesser importance to drinking water supplies (Nichols et al., 2004).

B.2.2.1 Sources and exposure

Mac organisms are natural inhabitants of water and soil environments (Falkinham, 2004). Water is considered the main reservoir (Percival et al., 2004; Vaerewijck et al., 2005), and the organisms can be encountered in natural aquatic systems worldwide, including marine waters and freshwater lakes, streams, ponds and springs (Falkinham, 2004; Percival et al., 2004). Mac organisms can be encountered in drinking water supplies, but generally in low numbers and at low frequency (Peters et al., 1995; Covert et al., 1999; Falkinham et al., 2001; Hilborn et al., 2006). However, Mac bacteria can survive in distribution system biofilms and grow there to reach significant populations (Falkinham et al., 2001). Counts of M. intracellulare in biofilms were observed to reach 600 colony-forming units (CFU) per square centimetre, on average (Falkinham et al., 2001). Feazel et al. (2009) observed that mycobacteria were enriched in plumbing system (showerhead) biofilms, reaching counts 100 times, based on higher numbers of genes being sequenced, above those in water samples. In another study, Tsintzou et al. (2000) observed a statistically significant decrease in the presence of environmental mycobacteria in drinking water samples after the replacement of the city's water distribution network. The authors attributed the reduction to the absence of distribution system biofilms (Tsintzou et al., 2000). Surveys of water samples from water treatment plants and residential dwellings have reported Mac isolation rates of 2-60% (von Reyn et al., 1993; Glover et al., 1994; Peters et al., 1995; Covert et al., 1999; Hilborn et al., 2006). Hilborn et al. (2006) recovered M. avium from roughly 50-60% of point-of-use samples (cold water taps) served by two water treatment plants. Concentrations ranged from 200 to > 300 CFU/500 mL (Hilborn et al., 2006). Von Reyn et al. (1993) isolated Mac organisms from 17-25% of water supply samples collected from hot water taps at patient care facilities (hospitals and clinics).

Other studies have reported a failure to isolate Mac organisms from water systems, instead detecting only other non-Mac mycobacteria (von Reyn et al., 1993; Le Dantec et al., 2002a; September et al., 2004; Sebakova et al., 2008). It has been suggested that the likelihood of exposure to Mac bacteria in water is diverse in various areas of the world, but, in general, may be less in developing countries (von Reyn et al., 1993; September et al., 2004). Mac organisms in biofilms have also been found in other human-made systems, such as cooling towers (Pagnier et al., 2009), ice machines (LaBombardi et al., 2002), nebulizer reservoirs, toilets and sinks (AWWA, 2006) and water meters (Falkinham et al., 2001). Studies have reported the isolation of non-tuberculous mycobacteria from groundwater, although M. avium has not been frequently detected (Falkinham et al., 2001; Vaerewijck et al., 2005).

Similar to Legionella, the growth and survival of Mac organisms can be enhanced by their ability to invade and survive in free-living amoebae, such as Acanthamoeba polyphaga or A. castellanii (Cirillo et al., 1997; Steinert et al., 1998). A key difference between Mac and Legionella, however, is that Mac organisms are able to replicate outside of amoebae, in biofilms (Steinert et al., 1998; Vaerewijck et al., 2005).

The ubiquitous nature of Mac organisms results from their ability to survive and grow under varied conditions. Mycobacteria can survive in water with few nutrients. Archuleta et al. (2002) observed that M. intracellulare was capable of surviving for over a year in reverse osmosis-deionized water. Mac organisms have also been shown to grow in natural waters over wide ranges of pH (5-7.5), salinity (0-2%) and temperature (10-51°C) (Sniadack et al., 1992; Falkinham et al., 2001). Water conditions that have been identified as being more favourable for the growth of Mac organisms include high levels of humic and fulvic acids, high zinc concentrations, low pH and low dissolved oxygen levels (Kirschner et al., 1992, 1999; Vaerewijck et al., 2005).

Infection through contact with M. avium and M. intracellulare has been well documented (Wendt et al., 1980; Grange, 1991; Glover et al., 1994; Montecalvo et al., 1994; von Reyn et al., 1994; Kahana et al., 1997; Aronson et al., 1999; Mangione et al., 2001). Inhalation of contaminated aerosols, during contact with contaminated hot tubs, spa pools or similar facilities, is most frequently cited as the route and source of infection (Kahana et al., 1997; Mangione et al., 2001; Rickman et al., 2002; Cappelluti et al., 2003; Lumb et al., 2004; Sood et al., 2007). Person-to-person transmission of the organisms is thought to be uncommon (Falkinham, 1996; Nichols et al., 2004). Evidence of the link between water supplies, particularly hot water supplies and Mac infection, has also been provided (von Reyn et al., 1994; Tobin-D'Angelo et al., 2004; Marras et al., 2005). Von Reyn et al. (1994) reported the detection of the same strain of M. avium in patients and hospital potable water supplies to which they had been exposed, but not in water supplies collected from patients' homes. Marras et al. (2005) documented a case of Mac-associated hypersensitivity pneumonitis where the patient strain was recovered from the shower and bathtub from the patient's home, but not the hot tub. Despite these links, it has been suggested that hospital and domestic drinking water-related cases represent a small proportion of Mac-related illness (von Reyn et al., 1994; Phillips and von Reyn, 2001). The infectious dose of Mac has not been well established. Rusin et al. (1997) proposed an oral infectious dose for mice of 104-107 organisms. True estimations of the inhaled infectious dose would be dependent upon (among other factors) the virulence of the organism and the immune status of the host.

B.2.2.2 Health effects

Mac organisms largely cause opportunistic infections in humans. Infections occur mostly in individuals who have weakened or suppressed immune systems (e.g., patients with acquired immunodeficiency syndrome [AIDS], the elderly or the very young) or persons with underlying respiratory conditions, such as cystic fibrosis. Mac-related disease rarely occurs in healthy people (Field et al., 2004). Mac organisms have low pathogenicity, so individuals can become colonized with the organisms without exhibiting any adverse health effects.

The main symptom of Mac lung infection is a chronic productive cough (cough with phlegm, saliva or mucus) (Field et al., 2004). Other symptoms can include fever, night sweats, fatigue and weight loss (Percival et al., 2004). However, it has been suggested that the secondary symptoms are less common unless the individual has extensive lung disease (Crow et al., 1957; Field et al., 2004). In those individuals with human immunodeficiency virus (HIV) or AIDS, Mac infection can spread to other parts of the body, including joints, skin, blood, liver and brain; the disease can be debilitating and life-threatening for these patients (Percival et al., 2004).

The true prevalence of Mac infections is not known, as it is not a reportable illness in Canada or the United States. Estimates of the rate of Mac-related pulmonary disease in the United States range from 1-2 cases to 5 cases per 100 000 persons per year, based on epidemiological studies conducted in various U.S. cities (Marras and Daley, 2002). Marras et al. (2007) estimated the prevalence of pulmonary non-tuberculous mycobacteria in Ontario to range from 9 to 14 positive isolations per 100 000 population over the years from 1997 to 2003. The authors further reported that, overall, Mac organisms were isolated in roughly 60% of the cases.

Mac diseases are treatable, but the clearing of these infections can be difficult, and treatment can have a high rate of failure (Field et al., 2004). Mycobacteria have demonstrated strong resistance to antimicrobial agents (Daley and Griffith, 2010). Antibiotics are delivered at high doses and often require a long administration period (e.g., several months to over a year) (Percival et al., 2004; Daley and Griffith, 2010).

B.2.2.3 Treatment technology

Water treatment technologies commonly used, including chemical disinfection and physical removal methods, have been tested for their ability to inactivate or remove mycobacteria from water supplies. Of these technologies, the most effective has been physical removal using conventional filtration (i.e., coagulation, flocculation and sedimentation). In one study, Falkinham et al. (2001) observed that water treatment plants treating surface water sources reduced mycobacteria numbers by 2-4 log through filtration and primary disinfection. A significant association between the frequency of detection of M. avium and high raw water turbidity was also reported. The authors were careful to note that reducing turbidity could represent one approach to reducing mycobacteria in drinking water, but that this procedure alone may not be completely sufficient to eliminate M. avium from the distribution system (Falkinham et al., 2001). It is important to note that even with good removal of organisms from the source water, the number of Mac organisms may increase in the distribution system (Falkinham et al., 2001). Mac organisms are more resistant than other microorganisms to commonly used disinfectants. The high concentration of mycolic acid and the hydrophobic surface characteristics of mycobacteria are primarily responsible for their high resistance to chemical disinfection (LeChevallier, 2004).

In a study to evaluate the change in the microbiological population of a water distribution system by changing the secondary disinfectant from chlorine to chloramine, the authors reported the presence of mycobacteria from sites where the chlorine residual was above 3 mg/L. In this same study, samples from the distribution system and at the point of use (i.e., showerheads) were analyzed, and the authors reported that the colonization rate of mycobacteria in samples from the distribution system and showerheads increased when the disinfectant was changed from chlorine to chloramine (Pryor et al., 2004).

For chlorination, Le Dantec et al. (2002b) reported varying chlorine sensitivities among a collection of various mycobacteria isolated from the distribution system (note: Mac organisms were not isolated in this study). The authors calculated that a CT value of 60 mg·min/L (e.g., 0.5 mg/L for 2 hours) would result in a 1.5-4 log reduction for environmental mycobacteria. R.H. Taylor et al. (2000) provided data on the susceptibility of environmental and patient isolates of M. avium to various disinfectants: chlorine, monochloramine, ozone and chlorine dioxide. The mean CT99.9 values (i.e., the CT values for a 3 log reduction) for the individual disinfectants were 51-204 mg·min/L for chlorine, 91-1710 mg·min/L for monochloramine, 0.10-0.17 mg·min/L for ozone and 2-11 mg·min/L for chlorine dioxide. The authors did note that there was significant variation in the susceptibility of different strains (R.H. Taylor et al., 2000).

In another study using chlorine dioxide, Vicuña-Reyes et al. (2008) reported CT99.9 values ranging from 3 to 36 mg·min/L (5-30°C), prompting the authors to conclude that the disinfectant can be effective in controlling mycobacteria. Compared with the CT values necessary to inactivate E. coli, the CT values necessary for inactivation of Mac have been reported to range from near equivalency to a few times greater (monochloramine); to tens to hundreds of times greater (ozone, chlorine dioxide); to over 2000 times greater (chlorine) (R.H. Taylor et al., 2000). Data have been provided suggesting that mycobacteria are more sensitive than Cryptosporidium oocysts to chlorine, monochloramine, chlorine dioxide and ozone and are as sensitive as or more sensitive than Giardia to all of these, with the exception of free chlorine (Jacangelo et al., 2002; LeChevallier, 2004).

In UV disinfection studies, Hayes et al. (2008) demonstrated that patient and environmental strains of M. avium and M. intracellulare exhibited a greater than 4 log reduction at UV fluences less than 20 mJ/cm2. The authors concluded that Mac organisms in free suspension could be readily inactivated by UV doses commonly employed in drinking water treatment (Hayes et al., 2008). LeChevallier (2004) reported that UV values required to inactivate mycobacteria are in the range of those required for other vegetative bacteria.

As stated above, there may be an increase of Mac organisms in the distribution system relative to levels leaving the treatment plant. By residing within biofilms or free-living amoebae, Mac organisms can further increase their resistance to inactivation. Steed and Falkinham (2006) observed that M. avium and M. intracellulare cells in biofilms were up to 1.8-4 times more resistant than cells in free suspension when exposed to chlorine. As with Legionella, successful control of Mac organisms requires control of the free-living amoebae and biofilms that support their persistence.

Mac organisms have also demonstrated resistance to elevated temperatures. Several authors have reported recovery of M. avium from hot water systems at temperatures between 50°C and 57°C (du Moulin et al., 1988; von Reyn et al., 1994; Covert et al., 1999; Norton et al., 2004). Additional factors thought to play a role in encouraging growth in the distribution system include high assimilable organic carbon levels, as well as distribution system materials and construction (e.g., pipe materials, gaskets, coatings, corroded pipes, dead ends, spaces, long storage times) (Falkinham et al., 2001). Similar to the distribution system control strategies described for Legionella (Bartram et al., 2007; Bentham et al., 2007), temperature control; control of water system design and construction to prevent the accumulation of biofilms, sediments or deposits; and nutrient control strategies should also prove effective in the control of Mac organisms.

B.2.2.4 Assessment

No suitable indicators have been identified to signal increasing concentrations of Mac organisms in water systems. For example, studies have found no relationship between the numbers of non-tuberculous mycobacteria recovered from reservoir water and coliform counts, HPC and total and free chlorine levels (Glover et al., 1994; Aronson et al., 1999). There is some evidence that M. avium presence is associated with turbidity in raw waters (Falkinham et al., 2001), but further exploration of this issue is needed.

Currently, the presence of mycobacteria in water is not regulated by any country or international organization, including Canada. The U.S. EPA has identified M. avium and M. intracellulare as waterborne health-related microbes that need additional research on their health effects, their occurrence in water and their susceptibility to treatment methods. These organisms have also been included in a list of candidate contaminants for possible regulation by the U.S. EPA (2009). At the present time, there is not sufficient information to warrant actions based on the presence of the organisms in the absence of disease.

B.2.3 Aeromonas

The genus Aeromonas has gained public health recognition as including organisms that can cause opportunistic infections in humans. Species of Aeromonas have been associated with gastroenteritis; however, understanding of the role that the organisms play in causing diarrhoeal illness is currently incomplete. Skin, wound and soft tissue infections with Aeromonas species as a result of exposure to contaminated water in non-drinking water scenarios have been well documented. It is believed that drinking water has the potential to serve as a route of transmission, but direct evidence of Aeromonas as a cause of drinking water-acquired gastrointestinal illness is lacking.

Aeromonads are Gram-negative, short, rod-shaped bacteria that share some similarities with Vibrio and E. coli. They are universally found, occurring naturally in virtually all water types. The genus Aeromonas contains more than 17 distinct genetic species. Three species--A. hydrophila, A. veronii biovar sobria (syn. A. sobria) and A. caviae--account for roughly 85% of human infections and are therefore considered to be the species of most importance for drinking water systems (Janda and Abbott, 1998, 2010).

B.2.3.1 Sources and exposure

Aeromonas species can be found in virtually all surface water types (freshwater, marine and estuarine) in all but the most extreme conditions of pH, salinity and temperature (Percival et al., 2004; AWWA, 2006). They are less frequently detected in groundwater, with their presence in these systems typically indicating well contamination (Havelaar et al., 1990; Massa et al., 1999 Borchardt et al., 2003).

Aeromonads are recognized animal pathogens (Percival et al., 2004; AWWA, 2006). The organisms have been isolated from the gastrointestinal tracts and infected tissues of a number of cold-blooded and warm-blooded animals, most notably fish, birds, reptiles and domestic livestock (U.S. EPA, 2006; Janda and Abbott, 2010). They have also been recovered from retail food items, such as meat, poultry and dairy products (Janda and Abbott, 2010). It has been suggested that animals may be an environmental reservoir for Aeromonas (Janda and Abbott, 2010).

The organisms are not considered to be natural faecal pathogens (U.S. EPA, 2006). Aeromonas species are not normally found in human faeces in high numbers (Janda and Abbott, 2010); however, a small percentage of the population can carry the bacteria in their intestinal tracts without showing symptoms of disease (von Graevenitz, 2007). The prevalence of Aeromonas in human faecal samples worldwide has been roughly estimated to be 0-4% for asymptomatic persons and as high as 11% for persons with diarrhoeal illness (Burke et al., 1983; U.S. EPA, 2006; von Graevenitz, 2007; Khajanchi et al., 2010). Individual studies have observed rates as high as 27.5% and 52.4% for asymptomatic persons and diarrhoeal illness cases, respectively (Pazzaglia et al., 1990, 1991). Numbers of Aeromonas are much higher in sewage, with concentrations greater than 108 CFU/mL having been reported (Percival et al., 2004).

Levels of Aeromonas in clean rivers, lakes and storage reservoirs have generally been reported to be in the range of 1-102 CFU/mL (Holmes et al., 1996). Aeromonas concentrations in surface waters receiving sewage contamination and nutrient-rich waters in the warmer summer months may reach 103-105 CFU/mL (Holmes et al., 1996; U.S. EPA, 2006). Groundwaters generally contain less than 1 CFU/mL (Holmes et al., 1996). Drinking water immediately leaving the treatment plant typically contains concentrations in the range of < 1-102 CFU/mL (Holmes et al., 1996; U.S. EPA, 2006; Pablos et al., 2009; Janda and Abbott, 2010) , with potentially higher concentrations in drinking water distribution systems (Payment et al., 1988; Chauret et al., 2001; U.S. EPA, 2006). Concentrations in individual environments can be expected to vary; however, the organisms can survive over wide ranges of pH (5-10) and temperature (2-42°C) (Percival et al., 2004). Water temperature is particularly important to Aeromonas growth. In temperate climes during the warmer months of the year, the bacteria have been shown to be more readily detected in source waters and water distribution systems (Chauret et al., 2001; U.S. EPA, 2006; Janda and Abbott, 2010). Aeromonads are also very versatile nutritionally. They are capable of growing to elevated numbers in water with high organic content and can also survive in low-nutrient waters (Kersters et al., 1996).

Similar to other bacteria, Aeromonas species can enter into a viable, non-culturable state under stressful conditions in aquatic environments. There is some debate at to what effect this state has on a species' viability and pathogenicity. Maalej et al. (2004) reported that cells of a strain of A. hydrophila rendered non-culturable under marine stress conditions lost their haemolytic and cytotoxic properties, but that these could be regained following recovery at warmer temperatures. In contrast, Mary et al. (2002) observed that viable, non-culturable cells of A. hydrophila lost their viability and that this could not be regained following a temperature upshift to 25°C. It has been suggested that survival properties may differ depending on the species and strain of Aeromonas (Brandi et al., 1999; Mary et al., 2002).

The organisms have been detected in the distribution systems of chlorinated drinking water supplies worldwide (Chauret et al., 2001; Emekdas et al., 2006; Långmark et al., 2007; September et al., 2007). As with other bacterial pathogens, the formation of biofilms and the presence of free-living amoebae have been identified as factors contributing to higher concentrations of Aeromonas encountered in drinking water distribution systems relative to finished water (September et al., 2007; Rahman et al., 2008). During an assessment conducted as part of their Unregulated Contaminant Monitoring Regulations, the U.S. EPA (2002) provided data indicating that Aeromonas could be detected in 11% of municipal systems serving more than 10 000 persons and 14% of systems serving fewer than 10 000 persons. The concentrations of Aeromonas reported were less than 10 CFU/100 mL in 78% of the samples (U.S. EPA, 2002). Limited studies have been conducted on Aeromonas-protozoa interactions within municipal supplies. Rahman et al. (2008) observed that the bacteria may use the free-living amoeba Acanthamoeba as a reservoir to improve transmission and for protection from disinfectants.

Exposure to Aeromonas species through direct contact of wounds or skin follicles with contaminated waters has been reported for recreational-type water environments, such as lakes, rivers, swimming pools and hot tubs (Gold and Salit, 1993; Manresa et al., 2009). Unusual water situations brought about by floods or disaster events can be expected to create similar opportunities for Aeromonas exposure. Wound infection with species of Aeromonas was a problem among victims of the tsunami in Thailand as a result of exposure to contaminated floodwaters (Hiransuthikul et al., 2005). Exposure to Aeromonas in contaminated floodwaters was also expected among victims and rescue workers following Hurricane Katrina (Presley et al., 2006). Person-to-person transmission of Aeromonas resulting in infection is not expected to occur (U.S. EPA, 2006).

The evidence for acquiring Aeromonas infection through the ingestion of drinking water is not well established, and this route of transmission is the subject of some debate (von Graevenitz, 2007). The presence of Aeromonas in finished drinking water supplies and distribution samples has been well documented, suggesting a possible route of transmission (LeChevallier et al., 1980; Payment et al., 1988; Kuhn et al., 1997; Borchardt et al., 2003; Emekdas et al., 2006; de Oliveira Scoaris et al., 2008). However, other findings have been cited that oppose this suggestion. Epidemiological investigations have demonstrated little evidence of direct connections between patient isolates of A. hydrophila and isolates recovered from their drinking water supplies. Borchardt et al. (2003) observed that Aeromonas isolates were infrequently found in stool samples of gastroenteritis patients and that those detected were not genetically related to isolates recovered from drinking water. Additionally, researchers have cited the virtual absence of reported outbreaks of diarrhoea against the near-universal presence of Aeromonas in water environments as evidence supporting the transmission of these organisms by a mechanism other than through drinking water (von Graevenitz, 2007; Janda and Abbott, 2010). Some researchers have speculated that for many faecal isolates of Aeromonas, colonization of the human gastrointestinal tract may only be fleeting (Janda and Abbott, 2010).

The ingested dose of Aeromonas necessary to cause gastrointestinal infections is uncertain. Limited study has suggested that a high dose is required (U.S. EPA, 2006; Janda and Abbott, 2010). In an early volunteer feeding study, Morgan et al. (1985) reported that only 2 of 57 individuals developed diarrhoea following ingestion of A. hydrophila strains at doses of up to 1010 CFU. It has been speculated that the concentrations required to cause illness are much higher than the numbers that would typically be found in treated drinking water supplies (U.S. EPA, 2006).

Recently, in a large survey of clinical and waterborne strains of Aeromonas collected from across the United States and worldwide, Khajanchi et al. (2010) reported detecting three isolates belonging to the A. caviae group that were genetically indistinguishable and possessed the same virulence factors. The authors suggested that these findings provided the first evidence of human infection and colonization by a waterborne Aeromonas strain.

B.2.3.2 Health effects

Aeromonas-associated diarrhoea has been encountered worldwide, mostly in normally healthy persons across all age groups (Janda and Abbott, 2010). Having low stomach acidity, receiving antimicrobial therapy and having compromised immune function (e.g., from HIV infection or through underlying disease, especially liver disease) are thought to be associated risk factors (Merino et al., 1995; Percival et al., 2004; von Graevenitz, 2007; Janda and Abbott, 2010). The association between Aeromonas and gastrointestinal illness is controversial (von Graevenitz, 2007; Janda and Abbott, 2010). Case reports and a small number of foodborne outbreaks have linked the presence of Aeromonas to cases of diarrhoeal disease (U.S. EPA, 2006; Janda and Abbott, 2010). However, at present, no outbreaks of gastrointestinal illness have been reported for which a strain of Aeromonas has been definitely identified as the causative agent (Janda and Abbott, 2010). Furthermore, researchers have been unable to find an animal model in which Aeromonas-mediated gastrointestinal illness can be replicated (U.S. EPA, 2006; Janda and Abbott, 2010).

Where Aeromonas species have been associated with gastroenteritis, the most common symptom is watery diarrhoea, accompanied by fever and abdominal pain (Janda and Abbott, 2010). Far less commonly, Aeromonas has been identified in association with other forms of gastrointestinal illness, ranging from a dysenteric type of illness with bloody stools to a chronic or subacute watery diarrhoea (Janda and Abbott, 2010). Aeromonas infections can also be asymptomatic, with individuals shedding the bacteria in their stools, but not showing any symptoms of disease (Percival et al., 2004).

Aeromonas species have been positively isolated from skin, wound and soft tissue infections (Percival et al., 2004; Janda and Abbott, 2010). These can range in scope from mild irritations (e.g., pus-filled lesions) to cellulitis (inflammation below the skin) to, in extreme cases, necrotizing fasciitis (flesh-eating disease) (Janda and Abbott, 2010). These are often the result of trauma or penetrating injury from occupational or recreational water exposure and are generally seen more frequently in adults than in children. Aeromonas has also recently been implicated in respiratory infections. However, these have been rare and have largely been caused by near-drownings or aspirations of contaminated waters unrelated to drinking water supplies (Janda and Abbott, 2010)

Factors responsible for the pathogenicity and virulence of Aeromonas species or strains are poorly understood. A number of potential virulence components have been identified that would appear to enable the organisms to behave as human pathogens. These include components such as pili, fimbriae and flagella for attachment and colonization; external lipopolysaccharides, capsules or surface layers to assist in evading host defences; and toxins, haemolysins, proteases and other enzymes for causing damage to host cells (von Graevenitz, 2007; Janda and Abbott, 2010). Current studies have been unable to specifically pinpoint which combination of factors would make a strain of Aeromonas behave as an enteropathogen (Janda and Abbott, 2010). Research has identified that a known diarrhoea-causing strain of A. hydrophila possesses four prospective virulence factors: two haemolysins (Act and HlyA), a heat-stable enterotoxin (Ast) and a heat-labile enterotoxin (Alt) (Erova et al., 2008 Janda and Abbott, 2010). Despite such findings, the role and relative significance of each remain uncertain, as studies have also found these factors distributed among numerous clinical and environmental strains in different combinations (Erova et al., 2008 von Graevenitz, 2007; Castilho et al., 2009; Janda and Abbott, 2010). It has been proposed that only certain subsets of Aeromonas strains have the ability to cause disease (Janda and Abbott, 2010).

Aeromonas is not a reportable organism in North America or in most countries worldwide (Janda and Abbott, 2010; PHAC, 2010). Of the case reports or outbreaks of Aeromonas-related illness encountered in the literature, most have been tied to food, hospitals, travel or non-water environments, or their causes are unknown. At present, no epidemiological evidence has been provided linking an Aeromonas outbreak to ingestion, inhalation or skin contact with treated drinking water supplies (U.S. EPA, 2006; von Graevenitz, 2007; Janda and Abbott, 2010).

As Aeromonas-related gastrointestinal illness is mild and self-limiting, treatment for infection is generally not necessary. However, for other presentations of infection, antibiotic therapy is usually implemented. Aeromonads are resistant to ampicillin and a variety of other ß-lactam antibiotics, including penicillin and some cephalosporins (Percival et al., 2004; Janda and Abbott, 2010).

B.2.3.3 Treatment technology

As mentioned previously, aeromonads are ubiquitous in many water environments. Consequently, they will be present in most source waters used for drinking water production. Nonetheless, existing evidence indicates that current treatment and disinfection methods can effectively remove Aeromonas from drinking water. Data from pilot-scale (Harrington et al., 2003; Xagoraraki et al., 2004) and full-scale (Chauret et al., 2001; El-Taweel and Shaban, 2001; Yu et al., 2008) investigations have demonstrated that well-operated conventional filtration systems (i.e., coagulation, flocculation and sedimentation) are capable of Aeromonas removals of up to 4 log. In a pilot-scale conventional treatment study, Xagoraraki et al. (2004) observed that reducing filter effluent turbidity to less than 0.2 NTU resulted in A. hydrophila removals of > 3 log to just under 4 log (median: 3.5 log). Yu et al. (2008) investigated the effectiveness of different water treatment processes in removing Aeromonas as measured using both culture-based and real-time polymerase chain reaction (PCR) detection methods. Conventional filtration (three full-scale plants) resulted in removals of culturable Aeromonas ranging from > 0.3 log to 4 log (Yu et al., 2008). The authors further reported that no culturable Aeromonas could be detected after sedimentation. Log removals as measured by real-time PCR detection correlated well with, but were routinely lower than, those demonstrated by the culture-based detection method (Yu et al. 2008).

For slow sand filtration, the authors examined one pilot-scale and two full-scale plants, reporting log removals of > 1 log (> 1 log for the pilot-scale plant and > 1.8 log for the full-scale plants). Culturable Aeromonas was not detected in samples collected post-filtration (Yu et al., 2008). Meheus and Peeters (1989) reported similar results for slow sand filtration, observing Aeromonas removals of 98-100%.

With membrane filtration, a full-scale plant included in the Yu et al. (2008) study demonstrated a capability of removing culturable Aeromonas by > 3.8 log.

Aeromonads are susceptible to inactivation by disinfectants commonly used in drinking water treatment, such as chlorine, monochloramine, chlorine dioxide, ozone and UV (Knøchel, 1991; Medema et al., 1991; Sisti et al., 1998; U.S. EPA, 2002, 2006). For chlorination, Sisti et al. (1998) reported Aeromonas T95 values of 5 minutes at a free chlorine concentration of 0.6 mg/L and 68 minutes at a free chlorine concentration of 0.05 mg/L in a laboratory-scale chlorination experiment. The authors also found Aeromonas (clinical strains) to be more susceptible to chlorine than E. coli (clinical strains). Free chlorine concentrations of 0.14 mg/L (10°C) and > 0.5 mg/L (20-37°C) were sufficient to produce a 5 log inactivation of clinical and nosocomial strains of Aeromonas within 5 minutes in an experiment conducted by Chamorey et al. (1999). In contrast, de Oliveira Scoaris et al. (2008) observed that the majority of Aeromonas strains (water and culture collection strains) were not killed after 1 minute of exposure to free chlorine at 1.2 mg/L.

Chauret et al. (2001) conducted a study at both full scale and pilot scale simultaneously to assess the presence of Aeromonas in source water and at various sites within the treatment plant and distribution system and to assess biofilm formation. The authors noted no detectable Aeromonas in treated water immediately after secondary disinfection with chloramine (dose range: 2-3 mg/L), despite observing counts ranging from < 1 to 490 CFU/100 mL after chlorine disinfection (pre-filtration) and post-granular activated carbon filtration.

With chlorine dioxide, Medema et al. (1991) reported CT99 values of 0.04-0.14 mg·min/L for a drinking water strain of A. hydrophila. In the same study, a naturally occurring Aeromonas population (predominantly A. sobria) was observed to be slightly more sensitive, with a reported CT99 of 0.1 mg·min/L.

For UV disinfection, data produced by the U.S. EPA (2002) suggested the capability for a 1 and 2 log inactivation of A. hydrophila at doses of 3 and 8 mWs/cm2, respectively (equivalent to 3 and 8 mJ/cm2)--doses significantly less than those commonly employed in water treatment.

In the distribution system, maintaining an adequate disinfectant residual should provide control of Aeromonas in the finished water. The potential exists for Aeromonas to regrow in the distribution system, however. During a year-long survey of a major drinking water distribution system in Scotland, Gavriel et al. (1998) reported that although Aeromonas was not detected in water samples collected downstream from chlorination prior to the distribution network, it could occasionally be recovered from distribution samples, even at locations maintaining a substantial chlorine residual (> 0.2 mg/L). Similarly, other studies have demonstrated that Aeromonas could be detected in municipal distribution systems at locations having temperatures below 14°C and chlorine residuals above 0.2 mg/L (Chauret et al., 2001; Pablos et al., 2009).

Elimination of Aeromonas in the distribution system once the organisms become established in biofilms can be difficult (Holmes and Nicolls, 1995; Gavriel et al., 1998; Långmark et al., 2007). Aeromonads sequestered in biofilms resist disinfection and persist for long periods (U.S. EPA, 2006). Elements important for helping to control Aeromonas growth include limiting the number of organisms entering the distribution system through effective treatment, maintaining low water temperatures, providing appropriate free chlorine residuals, limiting the levels of organic carbon compounds and proper maintenance of the distribution system (WHO, 2010).

B.2.3.4 Assessment

Some studies have been undertaken to determine whether the indicators currently used in the drinking water industry, including E. coli, total coliforms and HPC, can be used as surrogates for the presence of Aeromonas. Several studies have showed no evidence of a relationship between Aeromonas incidence and coliforms, E. coli or HPC (Holmes et al., 1996; Gavriel et al., 1998; Fernández et al., 2000; Pablos et al., 2009). Although no direct correlation exists between Aeromonas populations and total HPC, the organisms do make up a portion of HPC bacteria found in water and are detected by HPC tests (Pablos et al., 2009). The Netherlands has established drinking water standards for A. hydrophila, consisting of a median value (over a 1-year period) of 20 CFU/100 mL in water leaving the treatment plant and a 90th percentile value (over a 1-year period) of 200 CFU/100 mL in distribution system water (van der Kooij, 2003; Pablos et al., 2009). These values have been based on an assessment of achievability and are motivated by a precautionary approach, rather than on the public health significance of their occurrence in drinking water (WHO, 2002).

Aeromonas is not considered to be an indicator of faecal contamination or treatment failure (U.S. EPA, 2002). The organisms have been proposed as a possible supplemental indicator of drinking water quality by relating to the presence of biofilm. Therefore, if there are significant increases in Aeromonas concentrations in a drinking water supply, this indicates a general deterioration of bacteriological quality.

When looking at the overall public health significance of A. hydrophila in drinking water, further epidemiological studies are needed for a better understanding of the relationship between Aeromonas illness and the presence of these organisms in drinking water. Based on the current evidence, treated drinking water likely represents a very low risk. It has been proposed that in comparison with other pathogens that can potentially be acquired through drinking water, Aeromonas is at the low end of the scale in terms of relative risk (Rusin et al., 1997; Janda and Abbott, 2010). Nevertheless, it is advisable to minimize Aeromonas levels in drinking water supplies as much as is practical until its public health significance has been fully investigated.

B.2.4 Helicobacter pylori

Helicobacter pylori is a recognized human pathogen that can colonize the stomach. The understanding of how this organism is spread is still quite limited; however, it is believed that there are a few routes of transmission, including through drinking water (Percival and Thomas, 2009). The majority of people infected with H. pylori are asymptomatic, and they may live their entire lives with the organism. However, more serious disorders, such as peptic ulcers or stomach cancer, can develop in a small percentage of cases.

Helicobacter are Gram-negative, motile, small curved rods that are closely related to Campylobacter. The organisms have two distinct forms, a spiral rod shape and a shorter coccoid form, which is taken on under conditions of stress. To date, the coccoid form has been found to be non-culturable. The genus Helicobacter contains at least 25 species, as determined by deoxyribonucleic acid (DNA) sequencing, of which H. pylori is the species of relevance for the water industry. Other Helicobacter species have been detected in humans that have been associated with gastric illness; however, these are not considered to be as prevalent as H. pylori.

B.2.4.1 Sources and exposure

The primary reservoir identified for H. pylori is the human stomach (Dunn et al., 1997; Brown, 2000). It has been suggested that some animals (i.e., cats, dogs, sheep, primate monkeys) can be infected by H. pylori, but the consensus at present is that they do not play a significant role as reservoirs in transmitting this organism to humans (Baele et al., 2009; Haesebrouck et al., 2009). Although H. pylori has been cultured from human faeces, its isolation from water using culture methods has not been successful to date (Percival and Thomas, 2009). It is believed that the spiral culturable form rapidly transforms into a viable, non-culturable state (coccoid form) in the water environment. This is thought to be a response to environmental stresses, including changes in temperature, nutrient availability and osmolarity (Adams et al., 2003; Percival and Thomas, 2009.

Exact details on the transmission of H. pylori remain unclear (Bellack et al., 2006). Based on epidemiological findings, a higher risk of H. pylori infection exists among persons of low economic status living in crowded conditions or unhygienic environments (Brown, 2000; Gomes and De Martinis, 2004). Transfer mechanisms that have been proposed include gastric-oral, oral-oral and faecal-oral (Percival and Thomas, 2009). Overall, it is speculated that person-to-person transfer is the most likely route of transmission (Brown, 2000). The fact that it has not yet been possible to culture viable Helicobacter from the water environment has raised questions regarding the possibility of waterborne transmission. Nevertheless, there has been significant evidence provided in support of water as an important source of infection. Molecular techniques (PCR, fluorescent in situ DNA hybridization) have been used to confirm the presence of H. pylori in natural waters (Hegarty et al., 1999; Sasaki et al., 1999; Horiuchi et al., 2001; Benson et al., 2004; Moreno et al., 2007). As well, in the laboratory, H. pylori has been shown to survive for periods ranging from days up to weeks in sterile river water, stream water, saline solution and distilled water at a wide variety of pH values and at temperatures ranging from 4°C to 25°C (West et al., 1992; Shahamat et al., 1993; Adams et al., 2003; Azevedo et al., 2008). As with Legionella and mycobacteria, evidence has been supplied that biofilms and free-living waterborne amoebae may provide environmental niches where H. pylori can persist (Park et al., 2001; Winiecka-Krusnell et al., 2002; Watson et al., 2004; Bragança et al., 2007).

Waterborne transmission has been suggested as an important source of infection in developing countries (Bellack et al., 2006). Supporting evidence has come from epidemiological studies showing that individuals consuming untreated or contaminated waters had a high risk of infection (Klein et al., 1991; Goodman et al., 1996; McKeown et al., 1999; Herbarth et al., 2001; Brown et al., 2002; Rolle-Kampczyk et al., 2004). There has been less evidence supporting the importance of waterborne transmission in developed countries (Percival and Thomas, 2009) owing to the difficulty in isolating H. pylori from drinking water with culturable methods. These difficulties in isolating the bacteria are due to changes in morphology, growth and metabolism when H. pylori is exposed to varying environments (Bode et al., 1993). However, the detection of H. pylori in drinking water distribution systems using molecular techniques suggests that it can still play an important role (Baker and Hegarty, 2001; Watson et al., 2004; Gião et al., 2008; Percival and Thomas, 2009). Additional research is required to provide further insight into the persistence, viability and associated risk of H. pylori in drinking water systems.

The infectious dose necessary for colonization of humans is not known. Results of challenge studies suggest that it is less than 104 cells and related to stomach pH (Solnick et al., 2001; Graham et al., 2004). However, given the high percentage of infected individuals among the population and the evidence from cases of accidental infection (e.g., from laboratory work, use of improperly maintained endoscopes), the dose could be much lower (Langenberg et al., 1990; Matysiak-Budnik et al., 1995).

B.2.4.2 Health effects

Human infection with H. pylori leads to gastritis, or inflammation of the stomach lining (Dunn et al., 1997; Kusters et al., 2006). The organism colonizes the human stomach, stimulating the immune system and inflammatory cells, and it is this response that brings about gastritis. In the majority of H. pylori infections, there are no obvious signs of disease (Kusters et al., 2006). It has been well established that infections with H. pylori are generally acquired during childhood, with a lower frequency of infection in adults (Ernst and Gold, 2000; Allaker et al., 2002). Further, infection, once established, is considered to be lifelong unless treatment is pursued (Blaser, 1992; Kusters et al., 2006). Helicobacter pylori is the primary cause of peptic ulcers (Kuipers et al., 1995). It has been estimated that 85-95% of ulcers are the result of infection with this organism (Kuipers et al., 1995). Carriage of H. pylori has also been recognized as an important risk factor for the development of gastric cancer (i.e., gastric lymphoma and adenocarcinoma) (Dunn et al., 1997; Pinto-Santini and Salama, 2005). Broad estimates of the risk of infected persons developing these advanced diseases have been put at 10-20% for peptic ulcers and 1-2% for gastric cancer (Ernst and Gold, 2000; Kusters et al., 2006).

Infection with H. pylori is treatable (Scott et al., 1998; Vakil and Megraud, 2007), and data from animal and human infection studies suggest that an H. pylori vaccine is possible (Graham et al., 2004; Del Guidice et al., 2009). This area of research is currently being explored.

B.2.4.3 Treatment technology

Similar to other bacteria, a proportion of the H. pylori present in the source water will be removed using physical methods, such as conventional filtration (i.e., coagulation, flocculation and sedimentation). Helicobacter pylori is also susceptible to disinfectants commonly used in drinking water treatment (e.g., chlorine, UV, ozone and monochloramine).

Literature regarding the disinfection of H. pylori is limited when compared with that available for other waterborne bacterial pathogens. Investigations are difficult because the cells of H. pylori become viable but non-culturable in the environment, and this form cannot be detected easily by regular culture methods (Moreno et al., 2007). With chlorination, data provided from the few reported studies suggest log reductions of culturable H. pylori cells ranging from 0.3 log at a chlorine concentration of 0.1 mg/L for 1 minute (Baker et al., 2002) to > 4 log at a chlorine concentration of 0.5 mg/L for 80 seconds (Johnson et al., 1997) to approximately 7 log at a chlorine concentration of 1 mg/L for 5 minutes (Moreno et al., 2007). Moreno et al. (2007) conducted research using a combination of direct viable count and fluorescent in situ DNA hybridization methods specifically to study the effects of chlorination on H. pylori cell viability. The researchers demonstrated that viable H. pylori cells could be detected after 3 hours of exposure to a chlorine concentration of 1.0 mg/L, but not after 24 hours of exposure.

The current body of research suggests that the CT provided by a conventional water treatment plant is sufficient to inactivate H. pylori in the finished water. However, if H. pylori does enter the distribution system, potentially through a break in treatment or infiltration into the system, disinfectant residuals maintained in the distribution system are probably insufficient for inactivation (Baker et al., 2002). Disinfectant CT99 values for H. pylori reported by Baker et al. (2002) were 0.24 mg/L min for ozone, 0.299 mg/L min for chlorine and 9.5 mg/L min for monochloramine. In terms of response to disinfection, compared with E. coli, Baker et al. (2002) reported that H. pylori was statistically more resistant to chlorine and ozone, but not to monochloramine. Other authors have similarly reported H. pylori having greater resistance to chlorine compared with E. coli (Johnson et al., 1997; Moreno et al., 2007). For UV disinfection, Hayes et al. (2006) reported a greater than 4 log inactivation of culturable H. pylori cells at fluences of less than 8 mJ/cm2.

Association with biofilms has also been shown to protect H. pylori from disinfectants, similar to other bacterial pathogens. Gião et al. (2010) observed that H. pylori cells (measured by peptide nucleic acid probe) remained viable for at least 26 days following exposure to chlorine at 0.2 and 1.2 mg/L. Also, in contrast to findings provided by other researchers, the authors observed that H. pylori cells in suspension did not lose culturability after 30 minutes of exposure to chlorine at an initial concentration of 1.2 mg/L (Gião et al., 2010). Successful distribution system control of Helicobacter would similarly be aided by management steps to reduce the formation of biofilm and the presence of free-living amoebae in this environment.

B.2.4.4 Assessment

Overall, the predominant transmission route for H. pylori seems to be situation dependent, with person-to-person transmission playing a key role in many circumstances. Water and food appear to be of lesser direct importance, but they can still play a significant role in situations with improper sanitation and lax hygiene

Much is still unknown regarding the ecology and behaviour of H. pylori in water systems. However, sufficient information has been provided to suggest that H. pylori can be regarded as a potential human pathogen with the potential for waterborne transmission. Illness associated with H. pylori infection is of a mild or benign nature in the majority of cases, and outbreaks of illness have not been linked to the presence of H. pylori in drinking water supplies. Further research is needed to provide clarity on such topics as its presence in source waters, its susceptibility to treatment and disinfection, and its overall significance for drinking water systems in Canada.

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