Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection

The validity of using indicator organisms (total and fecal coliforms, enterococci, Clostridium perfringens, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses, Cryptosporidium, and Giardia) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%; C. perfringens, 61%; F-specific coliphages, ~40%; and enteric viruses, 31%. Cryptosporidium oocysts and Giardia cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable Cryptosporidium, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for Giardia cysts, Cryptosporidium oocysts, infectious Cryptosporidium, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study to assess the microbial contamination of Mya arenaria clams from the north shore of the St Lawrence River estuary, (Québec, Canada)

The aims of the present study were to assess the microbial quality of Mya arenaria clams from the north shore of the St. Lawrence River estuary and to validate various microbial indicator microorganisms of bivalve mollusks contamination. Clams were collected from nine sites, including four harvesting sites closed by virtue of the Canadian Shellfish Sanitation Program (CSSP). Six contamination indicators (fecal coliforms, somatic coliphages, F-specific coliphages, fecal streptococci, Clostridium perfringens, and Escherichia coli) and four pathogens (Campylobacter sp., Cryptosporidium parvum, Giardia sp., and Salmonella sp.) were identified in the clams. Indicators sensibility, specificity and predictive values with respect to the presence of pathogens were calculated. Pathogenic microorganisms detection frequency in clams was important (92%). Globally, pathogens tend to be less frequently detected in opened harvesting sites (p = 0.086). Although the assessed indicators were not perfect, when F-specific coliphages are associated with E. coli or fecal coliforms, a good sensibility (62%-64%) and good positive predictive value (88%) with respect to the investigated pathogens are obtained.     

Giardia and Cryptosporidium removal from waste-water by a duckweed (Lemna gibba L.) covered pond

AIMS: To determine the ability of duckweed ponds used to treat domestic waste-water to remove Giardia and Cryptosporidium. METHODS AND RESULTS: The influent and effluent of a pond covered with duckweed with a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%, respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. There was a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts by the pond (P < 0.001). Influent turbidity and parasite removal were also significantly correlated (Cryptosporidium and turbidity, P=0.05; Giardia and turbidity, P=0.01). CONCLUSIONS: The larger organisms (parasites) probably settled to the bottom of the pond, while removal of smaller bacteria and coliphages in the pond was not as effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Duckweed ponds may play an important role in wetland systems for reduction of Giardia and Cryptosporidium.     

Comparative analysis of Cryptosporidium, Giardia and indicator bacteria during sewage sludge hygienization in various composting processes

AIMS: To evaluate the suitability of Clostridium perfringens, Escherichia coli and enterococci as indicator organisms for Cryptosporidium and Giardia in treated sludge. METHODS AND RESULTS: Occurrence of Cryptosporidium oocysts and Giardia cysts, detected and enumerated by direct immunofluorescence microscopy, were compared with counts of indicator bacteria during six different sewage sludge hygienization processes, including closed reactor and open windrow composting, and sludge sanitation by quicklime or peat addition. No statistical correlation existed between the counts of indicator bacteria, Cl. perfringens, E. coli, and enterococci and occurrence of Cryptosporidium or Giardia. In sludge end-products, Giardia cysts were detected more frequently than Cryptosporidium oocysts. SIGNIFICANCE OF THE STUDY: Direct analysis is the best method to confirm the presence of (oo)cysts in sludge.     

Removal of indicator bacteria, human enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater primary treatment facility

Pathogens and fecal indicator bacteria occurrence and removal were studied for a period of 6 months at the Montreal Urban Community wastewater treatment facility. With a capacity of about 7.6 million cubic metres per day (two billion U.S. gallons per day), it is the largest primary physico-chemical treatment plant in America. The plant discharges a nondisinfected effluent containing about 20 mg/L of suspended matter and 0.5 mg/L of total phosphorus on the basis of average annual concentrations. BDO5 (annual mean) is 75 mg/L before treatment and 32 mg/L after treatment. Samples were collected for a period of 6 months, and they demonstrated that the plant was not efficient at removing indicator bacteria and the pathogens tested. Fecal coliforms were the most numerous of the indicator bacteria and their removal averaged 25%. Fecal streptococci removal was 29%, while Escherichia coli removal was 12%. In untreated sewage, fecal coliforms, E. coli, and human enteric viruses were more numerous in summer and early autumn. Fecal streptococci counts remained relatively similar throughout the period. Clostridium perfringens removal averaged 51%. Giardia cysts levels were not markedly different throughout the study period, and 76% of the cysts were removed by treatment. Cryptosporidium oocyst counts were erratic, probably due to the methods, and removal was 27%. Human enteric viruses were detected in all samples of raw and treated wastewater with no removal observed (0%). Overall, the plant did not perform well for the removal of fecal indicator bacteria, human enteric viruses, or parasite cysts. Supplementary treatment and disinfection were recommended to protect public health. Various alternatives are being evaluated.     

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2?kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E.?coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G.?duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.     

Concentrations of pathogens and indicators in animal feces in the Sydney watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Infectious Cryptosporidium parvum Oocysts in Final Reclaimed Effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Coliphages and indicator bacteria in birds around Boston Harbor

Droppings from feral populations of pigeons, geese and herring gulls from the urban/suburban environment around Boston Harbor, MA, USA contained up to 10⁶ somatic coliphages, 10⁸ enterococci, 10⁹ thermotolerant coliforms and 10² F-specific coliphages per gram of feces. Somatic coliphages, enterococci and thermotolerant coliforms were common in the feces of all three kinds of birds but F-specific coliphages were found in droppings from only three of 32 gulls. Thus these sources of bacterial and viral indicators should be considered when dealing with the ecology of fecal pollution indicators. Moreover, microbial indicators of fecal or sewage pollution originating from bird droppings may be mistaken for indicators that come from humans. This may cause an overestimate of the hazard from human pathogens in water and confound attempts to locate sources of fecal or sewage pollution. Received 12 May 1998/ Accepted in revised form 15 July 1998     

Virus occurrence in municipal groundwater sources in Quebec, Canada

A 1 year study was undertaken on groundwater that was a source of drinking water in the province of Quebec, Canada. Twelve municipal wells (raw water) were sampled monthly during a 1 year period, for a total of 160 samples. Using historic data, the 12 sites were categorized into 3 groups: group A (no known contamination), group B (sporadically contaminated by total coliforms), and group C (historic and continuous contamination by total coliforms and (or) fecal coliforms). Bacterial indicators (total coliform, Escherichia coli, enteroccoci), viral indicators (somatic and male-specific coliphages), total culturable human enteric viruses, and noroviruses were analyzed at every sampling site. Total coliforms were the best indicator of microbial degradation, and coliform bacteria were always present at the same time as human enteric viruses. Two samples contained human enteric viruses but no fecal pollution indicators (E. coli, enterococci, or coliphages), suggesting the limited value of these microorganisms in predicting the presence of human enteric viruses in groundwater. Our results underline the value of historic data in assessing the vulnerability of a well on the basis of raw water quality and in detecting degradation of the source. This project allowed us to characterize the microbiologic and virologic quality of groundwater used as municipal drinking water sources in Quebec.     

Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.     

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fate of bacterial indicators,viruses and protozoan parasites in a wastewater multi-component treatment system

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.     

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.     

Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.