Opportunistic Pathogens in Drinking Water Distribution Systems-A Review.

In contrast to "frank" pathogens, like Salmonella entrocolitica, Shigella dysenteriae, and Vibrio cholerae, that always have a probability of disease, "opportunistic" pathogens are organisms that cause an infectious disease in a host with a weakened immune system and rarely in a healthy host. Historically, drinking water treatment has focused on control of frank pathogens, particularly those from human or animal sources (like Giardia lamblia, Cryptosporidium parvum, or Hepatitis A virus), but in recent years outbreaks from drinking water have increasingly been due to opportunistic pathogens. Characteristics of opportunistic pathogens that make them problematic for water treatment include: (1) they are normally present in aquatic environments, (2) they grow in biofilms that protect the bacteria from disinfectants, and (3) under appropriate conditions in drinking water systems (e.g., warm water, stagnation, low disinfectant levels, etc.), these bacteria can amplify to levels that can pose a public health risk. The three most common opportunistic pathogens in drinking water systems are Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. This report focuses on these organisms to provide information on their public health risk, occurrence in drinking water systems, susceptibility to various disinfectants, and other operational practices (like flushing and cleaning of pipes and storage tanks). In addition, information is provided on a group of nine other opportunistic pathogens that are less commonly found in drinking water systems, including Aeromonas hydrophila, Klebsiella pneumoniae, Serratia marcescens, Burkholderia pseudomallei, Acinetobacter baumannii, Stenotrophomonas maltophilia, Arcobacter butzleri, and several free-living amoebae including Naegleria fowleri and species of Acanthamoeba. The public health risk for these microbes in drinking water is still unclear, but in most cases, efforts to manage Legionella, mycobacteria, and Pseudomonas risks will also be effective for these other opportunistic pathogens. The approach to managing opportunistic pathogens in drinking water supplies focuses on controlling the growth of these organisms. Many of these microbes are normal inhabitants in biofilms in water, so the attention is less on eliminating these organisms from entering the system and more on managing their occurrence and concentrations in the pipe network. With anticipated warming trends associated with climate change, the factors that drive the growth of opportunistic pathogens in drinking water systems will likely increase. It is important, therefore, to evaluate treatment barriers and management activities for control of opportunistic pathogen risks. Controls for primary treatment, particularly for turbidity management and disinfection, should be reviewed to ensure adequacy for opportunistic pathogen control. However, the major focus for the utility's opportunistic pathogen risk reduction plan is the management of biological activity and biofilms in the distribution system. Factors that influence the growth of microbes (primarily in biofilms) in the distribution system include, temperature, disinfectant type and concentration, nutrient levels (measured as AOC or BDOC), stagnation, flushing of pipes and cleaning of storage tank sediments, and corrosion control. Pressure management and distribution system integrity are also important to the microbial quality of water but are related more to the intrusion of contaminants into the distribution system rather than directly related to microbial growth. Summarizing the identified risk from drinking water, the availability and quality of disinfection data for treatment, and guidelines or standards for control showed that adequate information is best available for management of L. pneumophila. For L. pneumophila, the risk for this organism has been clearly established from drinking water, cases have increased worldwide, and it is one of the most identified causes of drinking water outbreaks. Water management best practices (e.g., maintenance of a disinfectant residual throughout the distribution system, flushing and cleaning of sediments in pipelines and storage tanks, among others) have been shown to be effective for control of L. pneumophila in water supplies. In addition, there are well documented management guidelines available for the control of the organism in drinking water distribution systems. By comparison, management of risks for Mycobacteria from water are less clear than for L. pneumophila. Treatment of M. avium is difficult due to its resistance to disinfection, the tendency to form clumps, and attachment to surfaces in biofilms. Additionally, there are no guidelines for management of M. avium in drinking water, and one risk assessment study suggested a low risk of infection. The role of tap water in the transmission of the other opportunistic pathogens is less clear and, in many cases, actions to manage L. pneumophila (e.g., maintenance of a disinfectant residual, flushing, cleaning of storage tanks, etc.) will also be beneficial in helping to manage these organisms as well.

Microbial community similarity and dissimilarity inside and across full-scale activated sludge processes for simultaneous nitrification and denitrification.

Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2-2.5% in the oxidation ditches and 0.4-0.7% at the other plants. Heterotrophic nitrifying-aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4-1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.

Protecting wastewater workers from disease risks: Personal protective equipment guidelines.

The 2013-2016 Ebola epidemic revived concerns about infection risks to wastewater workers. Prior research has shown that wastewater can contain a variety of known and emerging pathogens and that wastewater workers are at increased risk of infectious illnesses. However, guidelines on using personal protective equipment (PPE) to decrease these risks are lacking. We engaged 34 wastewater utility personnel and public health experts to conduct a job safety analysis identifying tasks in which workers could be exposed to pathogens and to develop a PPE selection matrix for preventing those exposures. We identified 43 relevant job tasks. Recommended PPE ranges from durable gloves (all tasks) to safety glasses (24 tasks), Tyvek suits or coveralls (4 tasks), and respiratory protection (N95 mask or face mask, depending on the activity, 10 tasks). The PPE selection matrix can serve as a guide for protecting the 120,000 wastewater workers in the United States from known and emerging pathogens. PRACTITIONER POINTS: Wastewater workers are at increased risk of infectious illnesses. Policies to protect wastewater workers from these illnesses are lacking. We developed guidelines for use of personal protective equipment by wastewater workers to prevent exposure to infectious agents.

Drinking water microbiome assembly induced by water stagnation.

What happens to tap water when you are away from home? Day-to-day water stagnation in building plumbing can potentially result in water quality deterioration (e.g., lead release or pathogen proliferation), which is a major public health concern. However, little is known about the microbial ecosystem processes in plumbing systems, hindering the development of biological monitoring strategies. Here, we track tap water microbiome assembly in situ, showing that bacterial community composition changes rapidly from the city supply following ~6-day stagnation, along with an increase in cell count from 103 cells/mL to upwards of 7.8 × 105 cells/mL. Remarkably, bacterial community assembly was highly reproducible in this built environment system (median Spearman correlation between temporal replicates = 0.78). Using an island biogeography model, we show that neutral processes arising from the microbial communities in the city water supply (i.e., migration and demographic stochasticity) explained the island community composition in proximal pipes (Goodness-of-fit = 0.48), yet declined as water approached the faucet (Goodness-of-fit = 0.21). We developed a size-effect model to simulate this process, which indicated that pipe diameter drove these changes by mediating the kinetics of hypochlorite decay and cell detachment, affecting selection, migration, and demographic stochasticity. Our study challenges current water quality monitoring practice worldwide which ignore biological growth in plumbing, and suggests the island biogeography model as a useful framework to evaluate building water system quality.

Core-satellite populations and seasonality of water meter biofilms in a metropolitan drinking water distribution system.

Drinking water distribution systems (DWDSs) harbor the microorganisms in biofilms and suspended communities, yet the diversity and spatiotemporal distribution have been studied mainly in the suspended communities. This study examined the diversity of biofilms in an urban DWDS, its relationship with suspended communities and its dynamics. The studied DWDS in Urbana, Illinois received conventionally treated and disinfected water sourced from the groundwater. Over a 2-year span, biomass were sampled from household water meters (n=213) and tap water (n=20) to represent biofilm and suspended communities, respectively. A positive correlation between operational taxonomic unit (OTU) abundance and occupancy was observed. Examined under a 'core-satellite' model, the biofilm community comprised 31 core populations that encompassed 76.7% of total 16 S rRNA gene pyrosequences. The biofilm communities shared with the suspended community highly abundant and prevalent OTUs, which related to methano-/methylotrophs (i.e., Methylophilaceae and Methylococcaceae) and aerobic heterotrophs (Sphingomonadaceae and Comamonadaceae), yet differed by specific core populations and lower diversity and evenness. Multivariate tests indicated seasonality as the main contributor to community structure variation. This pattern was resilient to annual change and correlated to the cyclic fluctuations of core populations. The findings of a distinctive biofilm community assemblage and methano-/methyltrophic primary production provide critical insights for developing more targeted water quality monitoring programs and treatment strategies for groundwater-sourced drinking water systems.

Occurrence and Control of Legionella in Recycled Water Systems.

Legionella pneumophila is on the United States Environmental Protection Agency (USEPA) Candidate Contaminant list (CCL) as an important pathogen. It is commonly encountered in recycled water and is typically associated with amoeba, notably Naegleria fowleri (also on the CCL) and Acanthamoeba sp. No legionellosis outbreak has been linked to recycled water and it is important for the industry to proactively keep things that way. A review was conducted examine the occurrence of Legionella and its protozoa symbionts in recycled water with the aim of developing a risk management strategy. The review considered the intricate ecological relationships between Legionella and protozoa, methods for detecting both symbionts, and the efficacy of various disinfectants.

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency.

The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods.

Impact of MBR cleaning and breaching on passage of selected microorganisms and subsequent inactivation by free chlorine.

Membrane bioreactors (MBRs) produce better quality effluent compared to conventional treatment processes but they are still subjected to the same disinfection requirements as conventional processes by many regulatory agencies. A research study consisting of bench-, pilot- and full-scale studies was conducted to characterize effluents produced from an MBR system operating under routine and challenged conditions and to assess the disinfection requirements for these effluents. Membrane cleaning did not seem to pose a substantial risk with respect to passage of target microorganisms; however, the membrane under breached conditions (turbidity > 0.5 NTU) resulted in an increase in a total coliform bacterial concentration up to 8500 CFU/100 mL. Adenoviruses were always detected in MBR filtrate samples by PCR (method detection limit of 10(3) genome copies per 25 µL reaction) irrespective of the membrane cleaning or breaching status. Passage of MS-2 bacteriophage through a breached membrane was lower compared to total coliform bacteria potentially due to their lower densities in the mixed liquor. Despite an increase in microbial concentration, a free chlorine CT of 30 mg-min/L was sufficient to achieve greater than 5-log removal of seeded MS-2 bacteriophage and removal of total coliform bacteria at or below the method detection limit (2 CFU/100 mL) for samples with a filtrate turbidity of 1.0 NTU. If such lower CT were to be employed, a significant decrease in plant footprint and operational costs could be realized.

Characterization of effluent water qualities from satellite membrane bioreactor facilities.

Membrane bioreactors (MBRs) are often a preferred treatment technology for satellite water recycling facilities since they produce consistent effluent water quality with a small footprint and require little or no supervision. While the water quality produced from centralized MBRs has been widely reported, there is no study in the literature addressing the effluent quality from a broad range of satellite facilities. Thus, a study was conducted to characterize effluent water qualities produced by satellite MBRs with respect to organic, inorganic, physical and microbial parameters. Results from sampling 38 satellite MBR facilities across the U.S. demonstrated that 90% of these facilities produced nitrified (NH4-N <0.4 mg/L-N) effluents that have low organic carbon (TOC <8.1 mg/L), turbidities of <0.7 NTU, total coliform bacterial concentrations <100 CFU/100 mL and indigenous MS-2 bacteriophage concentrations <21 PFU/100 mL. Multiple sampling events from selected satellite facilities demonstrated process capability to consistently produce effluent with low concentrations of ammonia, TOC and turbidity. UV-254 transmittance values varied substantially during multiple sampling events indicating a need for attention in designing downstream UV disinfection systems. Although enteroviruses, rotaviruses and hepatitis A viruses (HAV) were absent in all samples, adenoviruses were detected in effluents of all nine MBR facilities sampled. The presence of Giardia cysts in filtrate samples of two of nine MBR facilities sampled demonstrated the need for an appropriate disinfection process at these facilities.

Microbial community dynamics of an urban drinking water distribution system subjected to phases of chloramination and chlorination treatments.

Water utilities in parts of the U.S. control microbial regrowth in drinking water distribution systems (DWDS) by alternating postdisinfection methods between chlorination and chloramination. To examine how this strategy influences drinking water microbial communities, an urban DWDS (population ≅ 40,000) with groundwater as the source water was studied for approximately 2 years. Water samples were collected at five locations in the network at different seasons and analyzed for their chemical and physical characteristics and for their microbial community composition and structure by examining the 16S rRNA gene via terminal restriction fragment length polymorphism and DNA pyrosequencing technology. Nonmetric multidimension scaling and canonical correspondence analysis of microbial community profiles could explain >57% of the variation. Clustering of samples based on disinfection types (free chlorine versus combined chlorine) and sampling time was observed to correlate to the shifts in microbial communities. Sampling location and water age (<21.2 h) had no apparent effects on the microbial compositions of samples from most time points. Microbial community analysis revealed that among major core populations, Cyanobacteria, Methylobacteriaceae, Sphingomonadaceae, and Xanthomonadaceae were more abundant in chlorinated water, and Methylophilaceae, Methylococcaceae, and Pseudomonadaceae were more abundant in chloraminated water. No correlation was observed with minor populations that were detected frequently (<0.1% of total pyrosequences), which were likely present in source water and survived through the treatment process. Transient microbial populations including Flavobacteriaceae and Clostridiaceae were also observed. Overall, reversible shifts in microbial communities were especially pronounced with chloramination, suggesting stronger selection of microbial populations from chloramines than chlorine.

Evaluation of methods for the extraction of DNA from drinking water distribution system biofilms.

While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories.

Managing risks from virus intrusion into water distribution systems due to pressure transients.

Low or negative pressure transients in water distribution systems, caused by unexpected events (e.g. power outages) or routine operation/maintenance activities, are usually brief and thus are rarely monitored or alarmed. Previous studies have shown connections between negative pressure events in water distribution systems and potential public health consequences. Using a quantitative microbial risk assessment (QMRA) model previously developed, various factors driving the risk of viral infection from intrusion were evaluated, including virus concentrations external to the distribution system, maintenance of a disinfectant residual, leak orifice sizes, the duration and the number of nodes drawing negative pressures. The most sensitive factors were the duration and the number of nodes drawing negative pressures, indicating that mitigation practices should be targeted to alleviate the severity of low/negative pressure transients. Maintaining a free chlorine residual of 0.2 mg/L or above is the last defense against the risk of viral infection due to negative pressure transients. Maintaining a chloramine residual did not appear to significantly reduce the risk. The effectiveness of ensuring separation distances from sewer mains to reduce the risk of infection may be system-specific. Leak detection/repair and cross-connection control should be prioritized in areas vulnerable to negative pressure transients.

Bioluminescence-based method for measuring assimilable organic carbon in pretreatment water for reverse osmosis membrane desalination.

A bioluminescence-based assimilable organic carbon (AOC) test was developed for determining the biological growth potential of seawater within the reverse osmosis desalination pretreatment process. The test uses Vibrio harveyi, a marine organism that exhibits constitutive luminescence and is nutritionally robust. AOC was measured in both a pilot plant and a full-scale desalination plant pretreatment.

Implications of organic carbon in the deterioration of water quality in reclaimed water distribution systems.

Changes in water quality in reclaimed water distribution systems are a major concern especially when considering the potential for growth of pathogenic microbes. A survey of 21 wastewater process configurations confirmed the high quality effluent produced using membrane bioreactor (MBR) technology, but suggests that other technologies can be operated to produce similar quality. Data from an intensive twelve-month sampling campaign in four reclaimed water utilities revealed the important trends for various organic carbon parameters including total organic carbon (TOC), biodegradable dissolved organic carbon (BDOC), and assimilable organic carbon (AOC). Of the four utilities, two were conventional wastewater treatment with open reservoir storage and two employed MBR technology with additional treatment including UV, ozone, and/or chlorine disinfection. Very high BDOC concentrations occurred in conventional systems, accounting for up to 50% of the TOC loading into the system. BDOC concentrations in two conventional plants averaged 1.4 and 6.3 mg/L and MBR plants averaged less than 0.6 mg/L BDOC. Although AOC showed wide variations, ranging from 100 to 2000 µg/L, the AOC concentrations in the conventional plants were typically 3-10 times higher than in the MBR systems. Pipe-loop studies designed to understand the impact of disinfection on the microbiology of reclaimed water in the distribution system revealed that chlorination will increase the level of biodegradable organic matter, thereby increasing the potential for microbial growth in the pipe network. This study concludes that biodegradable organic carbon is an important factor in the microbial quality and stability of reclaimed water and could impact the public health risk of reclaimed water at the point of use.

Pyrosequencing analysis of bacterial biofilm communities in water meters of a drinking water distribution system.

The applicability of 454 pyrosequencing to characterize bacterial biofilm communities from two water meters of a drinking water distribution system was assessed. Differences in bacterial diversity and composition were observed. A better understanding of the bacterial ecology of drinking water biofilms will allow for effective management of water quality in distribution systems.

Regrowth of potential opportunistic pathogens and algae in reclaimed-water distribution systems.

A study of the quality of reclaimed water in treated effluent, after storage, and at three points in the distribution system of four plants in California, Florida, Massachusetts, and New York was conducted for 1 year. The plants had different treatment processes (conventional versus membrane bioreactor), production capacities, and methods for storage of the water, and the intended end uses of the water were different. The analysis focused on the occurrence of indicator bacteria (heterotrophic bacteria, coliforms, Escherichia coli, and enterococci) and opportunistic pathogens (Aeromonas spp., enteropathogenic E. coli O157:H7, Legionella spp., Mycobacterium spp., and Pseudomonas spp.), as well as algae. Using immunological methods, E. coli O157:H7 was detected in the effluent of only one system, but it was not detected at the sampling points, suggesting that its survival in the system was poor. Although all of the treatment systems effectively reduced the levels of bacteria in the effluent, bacteria regrew in the reservoir and distribution systems because of the loss of residual disinfectant and high assimilable organic carbon levels. In the systems with open reservoirs, algal growth reduced the water quality by increasing the turbidity and accumulating at the end of the distribution system. Opportunistic pathogens, notably Aeromonas, Legionella, Mycobacterium, and Pseudomonas, occurred more frequently than indicator bacteria (enterococci, coliforms, and E. coli). The Mycobacterium spp. were very diverse and occurred most frequently in membrane bioreactor systems, and Mycobacterium cookii was identified more often than the other species. The public health risk associated with these opportunistic pathogens in reclaimed water is unknown. Collectively, our results show the need to develop best management practices for reclaimed water to control bacterial regrowth and degradation of water before it is utilized at the point of use.

Development and application of a bioluminescence-based test for assimilable organic carbon in reclaimed waters.

Assimilable organic carbon (AOC) is an important parameter governing the growth of heterotrophic bacteria in drinking water. Despite the recognition that variations in treatment practices (e.g., disinfection, coagulation, selection of filter media, and watershed protection) can have dramatic impacts on AOC levels in drinking water, few water utilities routinely measure AOC levels because of the difficulty of the method. To simplify the method, the Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX test bacteria were mutagenized by using luxCDABE operon fusion and inducible transposons to produce bioluminescent strains. The growth of these strains can easily be monitored with a programmable luminometer to determine the maximum cell yield via luminescence readings, and these values can be fitted to the classical Monod growth curve to determine bacterial growth kinetics and the maximum growth rate. Standard curves using acetate carbon (at concentrations ranging from 0 to 1,000 microg/liter) resulted in coefficients of determination (r(2)) between luminescence units and acetate carbon levels of 0.95 for P-17 and 0.89 for NOX. The bioluminescence test was used to monitor reclaimed water, in which average AOC levels range between 150 and 1,400 microg/liter acetate carbon equivalents. Comparison of the conventional AOC assay and the bioluminescent assay produced an r(2) of 0.92.

Inferences drawn from a risk assessment compared directly with a randomized trial of a home drinking water intervention.

Risk assessments and intervention trials have been used by the U.S. Environmental Protection Agency to estimate drinking water health risks. Seldom are both methods used concurrently. Between 2001 and 2003, illness data from a trial were collected simultaneously with exposure data, providing a unique opportunity to compare direct risk estimates of waterborne disease from the intervention trial with indirect estimates from a risk assessment. Comparing the group with water treatment (active) with that without water treatment (sham), the estimated annual attributable disease rate (cases per 10,000 persons per year) from the trial provided no evidence of a significantly elevated drinking water risk [attributable risk=-365 cases/year, sham minus active; 95% confidence interval (CI) , -2,555 to 1,825]. The predicted mean rate of disease per 10,000 persons per person-year from the risk assessment was 13.9 (2.5, 97.5 percentiles: 1.6, 37.7) assuming 4 log removal due to viral disinfection and 5.5 (2.5, 97.5 percentiles: 1.4, 19.2) assuming 6 log removal. Risk assessments are important under conditions of low risk when estimates are difficult to attain from trials. In particular, this assessment pointed toward the importance of attaining site-specific treatment data and the clear need for a better understanding of viral removal by disinfection. Trials provide direct risk estimates, and the upper confidence limit estimates, even if not statistically significant, are informative about possible upper estimates of likely risk. These differences suggest that conclusions about waterborne disease risk may be strengthened by the joint use of these two approaches. Key words: drinking water, gastrointestinal, intervention trial, microbial risk assessment, waterborne pathogens.

Riverbank filtration for control of microorganisms: results from field monitoring.

Microbial monitoring was conducted over a period of more than 1 year at three full-scale riverbank filtration (RBF) facilities, located in the United States along the Ohio, Missouri, and Wabash Rivers. Results of this study demonstrated the potential for RBF to provide substantial reductions in microorganism concentrations relative to the raw water sources. Cryptosporidium and Giardia were detected occasionally in the river waters but never in any of the well waters. Average concentrations and log reductions of Cryptosporidium and Giardia could not be accurately determined due to the low and variable concentrations in the river waters and the lack of detectable concentrations in the well waters. Average concentrations of aerobic and anaerobic spore-forming bacteria, which have both been proposed as potential surrogates for the protozoans, were reduced at the three facilities by 0.8 to > 3.1 logs and 0.4 to > 4.9 logs, respectively. Average concentrations of male-specific and somatic bacteriophage were reduced by > 2.1 logs and 3.2 logs, respectively. Total coliforms were rarely detected in the well waters, with 5.5 and 6.1 log reductions in average concentrations at the two wells at one of the sites relative to the river water. Average turbidity reductions upon RBF at the three sites were between 2.2 and 3.3 logs. Turbidity and microbial concentrations in the river waters generally tracked the river discharge; a similar relationship between the well water concentrations and river discharge was not observed, due to the low, relatively constant well water turbidities and lack of a significant number of detections of microorganisms in the well waters. Further research is needed to better understand the relationships among transport of pathogens (e.g., Cryptosporidium, Giardia, viruses) and potential surrogate parameters (including bacterial spores and bacteriophage) during RBF and the effects of water and sediment characteristics on removal efficiency.