Bacteriophages as indicators of fecal pollution and enteric virus removal

Abstract

Bacteriophages are an attractive alternative to fecal indicator bacteria (FIB), particularly as surrogates of enteric virus fate and transport, due to their closer morphological and biological properties. Based on a review of published data, we summarize densities of coliphages (F+ and somatic), Bacteroides spp. and enterococci bacteriophages (phages) in individual human waste, raw wastewater, ambient fresh and marine waters and removal through wastewater treatment processes utilizing traditional treatments. We also provide comparisons with FIB and enteric viruses whenever possible. Lastly, we examine fate and transport characteristics in the aquatic environment and provide an overview of the environmental factors affecting their survival. In summary, concentrations of bacteriophages in various sources were consistently lower than FIB, but more reflective of infectious enteric virus levels. Overall, our investigation indicates that bacteriophages may be adequate viral surrogates, especially in built systems, such as wastewater treatment plants.

Introduction

Due to intrinsic economic, aesthetic and recreational benefits, an estimated 39% of the United States population, and over 50% of the global population lives in close proximity to rivers, lakes or oceans (Kummu et al. 2011; National Oceanic and Atmospheric Administration 2013). Enteric pathogens can enter aquatic environments through wastewater treatment plants (WWTPs) effluents, industrial waste, concentrated animal feeding operations or by more imperceptible means such as undocumented septic system failures, leaking sewers, as well as from agricultural and wild animals. Human excreta, however, are the primary source of infectious enteric viruses of potential concern to recreators, due to high shedding rates of infected individuals (Osborne et al. 2015; Taniuchi et al. 2015; Sabria et al. 2016) and low dose required for illness (Atmar et al. 2014; Messner et al. 2014). Since enteric viruses are the leading etiologic agents of waterborne disease outbreaks in recreational water settings (Sinclair et al. 2009), exposure to human waste (e.g. untreated sewage, secondary disinfected wastewater effluent, primary wastewater effluent) represents the highest manageable pathogen risk to public health (Soller et al. 2010a; Soller et al. 2010b). While the health risk resulting from exposure to cattle manure may not be substantially different due to the presence of zoonotic pathogens, most other animal sources tend to be of lesser concern (Soller et al. 2010b; Soller et al. 2014).

Due to the difficulties associated with direct detection of pathogens in water, the potential presence of fecal pollution is typically assessed by using fecal indicator bacteria (FIB) which include, fecal coliforms, Escherichia coli and enterococci (Ashbolt 2001). The utility of culture-based assessments of FIB for use in protecting public health, however, has limitations. Since FIB reside in the gastrointestinal tract of many different animals, they cannot be used to distinguish between fecal contamination sources. FIB also display different fate and transport characteristics within waste treatment infrastructure and natural aquatic environments when compared to that of viral and protozoan pathogens (Ashbolt 2001; Boehm et al. 2009a). Bacteriophages are viruses that are dependent on a bacterial host for replication, and those infecting FIB or other commensal intestinal species (e.g. Bacteroides) are subsequently shed by hosts and follow similar routes of diffusion into the environment to that of enteric viral pathogens. Furthermore, bacteriophages have similar morphological characteristics to those of many enteric pathogenic viruses suggesting that they can better mimic their survival (Payment et al. 2001; Payment and Locas 2011). Therefore, bacteriophage have been considered potentially attractive surrogates of viral pathogens because of their similarity in persistence in both built environments (e.g. WWTPs) as well as recreational waters. In this review, we focus on E. coli, Bacteroides spp., and enterococci bacteriophages to explore their potential as fecal indicators.

Bacteriophages infecting E. coli, Bacteroides spp. and enterococci

Coliphages

Coliphages are viruses that have the ability to infect E. coli, and are split into two categories based on the route of bacterial host infection. Somatic coliphages attach through specific protein receptors found on the outer host cell membrane (Kott 1974), while male specific (F+) coliphages (F-RNA & F-DNA) infect via the host’s sex (F) pili (Davis 1961). They are the most extensively studied phages for considerations as possible indicators of fecal pollution and surrogates for viral fate and transport (Skraber et al. 2002; Cole et al. 2003; Duran et al. 2003; Skraber et al. 2004a) and are a part of multiple regulatory frameworks involving monitoring of groundwater (United States Environmental Protection Agency 2006), biosolids (Department of Environment and Conservation 2012), water recycling (Queensland Government Environmental Protection Agency 2005; North Carolina Environmental Quality 2011) and aquaculture practices (Food and Drug Administration and Interstate Shellfish Sanitation Commission 2015). In addition, coliphages are now being considered for monitoring of recreational waters (United States Environmental Protection Agency 2015; 2016). Please see recent reviews regarding coliphages as model organisms (Jofre et al. 2016) and possible indicators of fecal contamination (United States Environmental Protection Agency 2015).

Somatic coliphages are classified into four major taxonomic groups, Myoviridae (linear double-stranded DNA), Siphoviridae (linear double-stranded DNA), Podoviridae (linear double- stranded DNA), Microviridae (circular double-stranded DNA); with the F+ divided into Leviviridae (single-stranded RNA), Inoviridae (single-stranded DNA) and Tectiviridae (double-stranded DNA) (Lute et al. 2004; Mesquita et al. 2010). While somatic coliphages are typically detected using E. coli, they have also been known to also infect Klebsiella and Shigella spp. (Leclerc et al. 2000; Muniesa et al. 2003). Among the F+ coliphage families, F-DNA coliphages belonging to Inoviridae have received less attention as water quality indicators since they are not as abundant as F-RNA coliphages (Leviviridae), and morphologically are less similar to enteric viruses (Furuse 1987; Leclerc et al. 2000). Based on the nucleotide sequence similarities, F-RNA coliphages belonging to the family Leviviridae, are subdivided into two genera Levivirus and Allolevivirus (King et al. 2011). Considering serological properties, and physiochemical measurements, Levivirus is further subdivided into genogroups I and II, and Allolevivirus into III and IV (Vinje et al. 2004; Friedman et al. 2009). Further studies positively link certain genogroups to a specific source of fecal contamination; human excreta largely contain higher proportions of genogroups II and III, while non-human sources (bovine, swine and avian) predominantly, but not exclusively harbor genogroups I and IV (Griffin et al. 2000; Cole et al. 2003; Noble et al. 2003). However, association of F+ genotypes and fecal sources is imperfect as cross-reactivity has been recorded (Hsu et al. 1995; Stewart-Pullaro et al. 2006; Stewart et al. 2006; Wolf et al. 2010; Hartard et al. 2015).

Bacteroides spp. phages

Bacteroides are Gram-negative, obligate anaerobic, pleomorphic rod-shaped bacteria that are commonly found in high concentrations in both human and other animal feces (Harwood et al. 2014). Bacteriophages infecting Bacteroides have been extensively studied to investigate their potential for microbial source tracking (MST) (Gomez-Donate et al. 2011; Nnane et al. 2011; Harwood et al. 2013; Jofre et al. 2014; Diston et al. 2015; Venegas et al. 2015). Some Bacteroides spp. host strains (HSP40, RYC2056 and RYC4023) are useful in discriminating phages from different animal fecal sources (Gomez-Donate et al. 2011), however geographical limitations (Cornax et al. 1990; Grabow et al. 1995; Araujo et al. 1997), cross-reactivity with domestic animal fecal samples (Gomez-Donate et al. 2011), or otherwise low and highly variable detection rates (Tartera and Jofre 1987; Tartera et al. 1989) have prompted research into other more applicable Bacteroides spp. strains. B. thetaiotaomicron (GA17) and B. fragilis (GB124) have been identified as closely associated with human fecal pollution sources for MST purposes (Payan et al. 2005; Ebdon et al. 2012). Distribution of GA17 bacteriophages seems to be largely limited to Europe, although detectable levels have also been reported in Tunisia and Columbia (Blanch et al. 2006; Sirikanchana et al. 2014; Venegas et al. 2015; Yahya et al. 2015), while B. fragilis (GB124) seems to be somewhat more widespread, but suffers from lower concentrations in sewage and environmental waters (Ebdon et al. 2007; Nnane et al. 2011; Ebdon et al. 2012; Harwood et al. 2013; McMinn et al. 2014; Diston and Wicki 2015). Efforts in identifying new Bacteroides spp. bacteriophage hosts continue with the identification of strains that can potentially differentiate between human feces (ARABA 84, CA8) versus animal wastes (RBA 63 and KBA 60) (Wicki et al. 2011; Diston and Wicki 2015; Venegas et al. 2015).

Enterococci phages

Intestinal Enterococcus spp. are facultatively anaerobic, Gram-positive cocci that are abundant in both human and animal feces. Some species (e.g. E. faecalis and E. faecium) have been identified as being frequently found in human intestines, while members of other species (E. casseliflavus, E. mundtii and E. gallinarum) typically reside in non-human hosts (recently reviewed in (Byappanahalli et al. 2012)). This bacterial host strain association suggests that the resulting enterophages could be used for MST purposes (Bonilla et al. 2010; Santiago-Rodriguez et al. 2010; Santiago-Rodriguez et al. 2013). Studies have reported detection of E. faecalis phages in human feces, while absent from non-human sources, so understandably the majority of enterophage MST research has utilized this bacterial species (Bonilla et al. 2010; Santiago-Rodriguez et al. 2010; Purnell et al. 2011; Santiago-Rodriguez et al. 2013). Enterophages have been shown to have survival times similar to human enteric viruses in both fresh and marine waters, however, the majority of this research has been restricted to tropical and subtropical regions of the world (Bonilla et al. 2010; Santiago-Rodriguez et al. 2012; Santiago-Rodriguez et al. 2013). To determine the utility of enterophages as indicators of fecal pollution and enteric virus surrogates, future studies are needed outside this limited geographical area.

Indigenous bacteriophages in wastewater and environmental waters

Our literature search primarily focused on manuscripts (a minimum of two) that reported quantifiable bacteriophage densities rather than presence/absence or percentage data. If available, we also included bacterial indicator (FIB) and viral pathogen measurements (infectious enteric viruses, molecular signal for Adenoviruses and Noroviruses) when reported from the same samples used to generate bacteriophage data. Infectious enteric viruses in this context refers to those organisms that can be grown in continuous mammalian cell lines. The studies included here that reported on the infectious viruses predominantly relied on Buffalo Green Monkey (BGM) kidney cell line, although some also used rhabdo(myo)sarcoma, Human Caucasian Colon Adenocarcinoma (Caco-2) or African Green Monkey kidney (MA-104) lines. Data was collected from text, tables, supplemental materials or estimated from figures when not available otherwise. We collected the mean of each data set from which an overall mean was calculated per organism and/or matrix. In instances where there was no detectable data reported, we substituted it with a zero. Concentrations of all microorganisms were log₁₀ transformed and normalized to 100 mL, irrespective of how the data was originally reported. When necessary log₁₀ reduction values were determined using the following formula: log₁₀ reduction = log₁₀T₀−log₁₀T_N where T₀ is the concentration at the beginning of the wastewater treatment process and T_N is the concentration at the end of the treatment stage or the entire treatment train. Lastly, it is important to highlight various assumptions we employed when collecting the data. We assumed that all methods regardless of the protocol used or the intended microbial target perform equally well while also assuming that matrix (human and animal waste, wastewater, freshwater and marine water), geographic location and seasonality did not affect method performance. Because of these limitations we are unable to define any conclusive relationships in the gathered data, but focus instead on describing general trends we observed.

Our review included data from a total of 89 published manuscripts from five continents and 25 countries including Argentina, Austria, Canada, China, Columbia, Cyprus, Finland, France, Germany, Greece, Ireland, Israel, Italy, Japan, Malaysia, Netherlands, Singapore, South African Republic, South Korea, Spain, Sweden, Switzerland, Tunis, United Kingdom and United States. Occurrence data included values from human and non-human excreta, environmental waters as well as WWTP inactivation studies of FIB, coliphages (somatic and F+), B. fragilis bacteriophages (RYC2056, GB124, HSP40), B. thetaiotamicron bacteriophages (GA17), enterophages infecting E. faecalis and viral pathogens (infectious enteric viruses as well as Noroviruses and Adenoviruses quantified via qPCR).

Occurrence in feces and wastewaters

Human fecal sources

The focus of this section is to primarily review the concentrations of indigenous viral indicators, while including bacterial indicators as well as human viral pathogens measured in untreated wastewater. Our emphasis on untreated wastewater rather than human fecal matter and other on-site collection/disposal systems (e.g. septic tanks) was governed by the preponderance of wastewater data in the published literature. When available, studies indicate that concentrations of bacteriophage were typically lower in feces of individual humans and from on-site systems when compared to centralized infrastructure (such as WWTPs) (Calci et al. 1998; Lucena et al. 2003; Harwood et al. 2013; Diston and Wicki 2015). While standard methods are generally used for fecal indicator enumeration, typically little to no data was reported for recoveries from the matrices studied, hence adding uncertainties to reported concentrations.

Overall, concentrations of FIB were routinely higher compared to those of bacteriophages, while human viral pathogen levels tended to be the lowest (Figure 1). Of the FIB, mean fecal coliform concentrations (log₁₀ 6.84 ± 0.77 CFU/100 mL) were the highest and ranged from log₁₀ 4.74 −8.87 per 100 mL, followed by E. coli which ranged from log₁₀ 6.34−7.00 per 100 mL (mean log₁₀ 6.42 ± 0.63). Mean enterococci concentrations were the lowest of the three (log₁₀ 5.88 ± 0.55) and ranged from log₁₀ 4.4−6.91 per 100 mL (Figure 1).

Figure 1.

Mean concentrations and 95% confidence intervals of FIB and bacteriophages (expressed as CFU, PFU or MPN), as well as viral pathogens (expressed as MPN for infectious viruses or genomic copies for all others) in untreated wastewater. The size of the symbol is proportionate to the number of studies* (given in parentheses) used to derive the data.

*References: (Tartera et al. 1989; Calci et al. 1998; Chauret et al. 1999; Puig et al. 1999; Baggi et al. 2001; Quinonez-Diaz et al. 2001; Rose et al. 2001; Uemura et al. 2002; Alcaide et al. 2003; Lucena et al. 2003; Queralt et al. 2003; Veschetti et al. 2003; Lucena et al. 2004; Payan et al. 2005; Blanch et al. 2006; Haramoto et al. 2006; Ebdon et al. 2007; Zhang and Farahbakhsh 2007; Reinoso et al. 2008; Petrinca et al. 2009; Boczek et al. 2010; Bonilla et al. 2010; Fu et al. 2010; Santiago-Rodriguez et al. 2010; Vijayavel et al. 2010; Wu et al. 2010; Ebdon et al. 2012; Flannery et al. 2012; Francy et al. 2012; Muniesa et al. 2012a; Harwood et al. 2013; Pradhan et al. 2013; Santiago-Rodriguez et al. 2013; McMinn et al. 2014; Haramoto et al. 2015; Montazeri et al. 2015).

When bacteriophage concentrations were examined, both coliphage types (somatic and F+) were generally higher than any Bacteroides spp. or enterococci phages (Figure 1). The observed mean concentrations for somatic and F+ were log₁₀ 5.26 ± 0.96 (range: log₁₀ 1.25–8.00) and log₁₀ 5.24 ±0.92 (range: none-detected −log₁₀ 8.00) PFU per 100 mL, respectively (Figure 1); levels similar to those reported recently in a comprehensive systematic literature review (Eftim 2016). Of the Bacteroides spp. bacteriophages, RYC2056 type was the most numerous and it ranged from none- detected to log₁₀ 4.92 with an average of log₁₀ 4.05 ± 1.40 PFU per 100 mL. Levels of B. fragilis HSP40 bacteriophage followed with an average of log₁₀ 3.10 ± 1.49 (ranging from log₁₀ 1.60–4.65) PFU per 100 mL (Figure 1). The average of two remaining Bacteroides spp. phages were lower (log₁₀ 2.62 ± 2.25 and log₁₀ 2.61 ± 1.38) and ranged from no detection for both groups to log₁₀ 5.04 and log₁₀ 4.36 PFU per 100 mL for GA17 and GB124, respectively (Figure 1). It should be noted that GA17 and GB124 averages are likely to be affected by an uneven geographic distribution, most notably in the areas where they are not endemic. Bacteriophages infecting E. faecalis exhibited the lowest average concentration (log₁₀ 2.19 ± 0.52), ranging from log₁₀ 1.15 −2.89 PFU per 100 mL.

Levels of infectious human enteric viruses were clearly the lowest of all microorganisms examined and averaged log₁₀ 0.52 ± 0.82, with a range of log₁₀ 0.64–2.41 MPN per 100 mL of wastewater (Figure 1). Noting again, that recovery data was generally not provided, yet is known to be highly variable (Petterson et al. 2015). Human viral gene targets measured by qPCR methods were considerably higher compared to culturable infectious virus concentrations, averaging log₁₀ 3.84 ± 1.33 for Norovirus (combined average of genogroups I and II) and log₁₀ 2.54 ± 2.27 for Adenovirus (Figure 1). Norovirus levels are comparable to those recently reported in a systematic review and meta-analysis detailing Norovirus occurrence in raw sewage (Eftim et al. 2017).

Non-human fecal sources

This section focuses on bacteriophage data measured in animal solid waste as well as animal wastewater and/or fecal slurries. Animal solid waste included samples collected from 21 species belonging to ruminants (cattle, elk, sheep, deer, goat), non-ruminant herbivores (donkey, horse, rabbit), omnivores (baboons, gorillas, orangutans, pigs, vervet monkeys), carnivores (cat, dog) and birds (chicken, duck, goose, pigeon, seagull, turkey). Animal fecal slurries and wastewater represented a mixture of individual samples, in many instances from multiple animal species.

Similar to untreated municipal wastewater, somatic and F+ coliphages were present in the highest concentrations, along with B. fragilis RYC2056 phage. In animal fecal slurries and wastewater, average concentrations were log₁₀ 4.67 ± 0.96, 4.39 ± 0.28 and log₁₀ 3.61 ±1.89 per 100 mL for F+, RYC2056 and somatic coliphages, respectively (Hill and Sobsey 1998; Puig et al. 1999; Hill and Sobsey 2001; Hill et al. 2002; Payan et al. 2005; Blanch et al. 2006; Ebdon et al. 2007; Muniesa et al. 2012a; Harwood et al. 2013; McMinn et al. 2014).

In animal solid waste, somatic coliphages were higher (log₁₀ 2.55 ±2.05 per gram) compared to F+ (log₁₀ 1.29 ±1.32 per gram), but there was not enough data to compare for RYC2056 (Grabow et al. 1995; Calci et al. 1998; Miller et al. 1998; Muniesa et al. 1999; Yee et al. 2006; McMinn et al. 2014; Diston and Wicki 2015). The other Bacteroides spp. phages were mainly measured in slurry and wastewater and their concentrations ranged from log₁₀ 1.52 ± 1.95, log₁₀ 0.69 ± 0.80 and log₁₀ 0.23 ± 0.63 per 100 mL for GA17, GB124 and HSP40, respectively (Payan et al. 2005; Ebdon et al. 2007; Muniesa et al. 2012b; Harwood et al. 2013; McMinn et al. 2014; Diston and Wicki 2015). In addition, GB124 phage was not detected in any solids examined (McMinn et al. 2014; Diston and Wicki 2015). Enterophage was also not detected in any animal solid samples, but there was insufficient information for slurry and wastewater (Bonilla et al. 2010; Santiago-Rodriguez et al. 2010; Santiago-Rodriguez et al. 2013).

Removal through conventional municipal wastewater treatment processes

In this subsection, our interest was to describe microbial removal at full-scale wastewater treatment, and therefore we excluded all laboratory and bench scale studies. Due to paucity of the available data we pooled together all Bacteroides spp. phages into one group. Since there was not enough data to describe removals through individual stage(s) of the wastewater treatment train we pooled data and present it as log₁₀ reduction irrespective of any particular unit of operation (e.g. primary treatment, secondary treatment, disinfection). Even though there was uneven number of studies per organism, the distribution of papers describing removal through a particular unit of operation for each microorganism was relatively consistent. When grouped by the absence or presence of disinfection, the percentage of manuscripts belonging to each category was 54/47% (fecal coliforms), 63/37% (E. coli), 57/43% (enterococci), 74/26% (somatic coliphages), 71/29% (F+ coliphages), 68/32% (Bacteroides spp. bacteriophages), 50/50% (Adenoviruses), 44/56% (Nororvirus) and 81/29% (infectious enteric viruses). The pooled data was subjected to Kruskal-Wallis analysis of variance (ANOVA) with Dunn’s post-hoc test performed using Sigma Plot version 13.0 (Systat Software, San Jose, CA) to determine whether statistically significant difference (P > 0.05) exists in log₁₀ reductions among different organisms (Table 1).

Table 1.

Kruskal-Wallis ANOVA with Dunn’s post-hoc tests on log₁₀ reduction values through various wastewater treatment processes for bacterial and viral indicator organisms and pathogens obtained from published literature

FC (38)	E. coli (32)	Enterococci (36)	SOMPH (111)	F+PH (102)	BSPH (22)	Adenovirus (10)	Norovirus (28)	Inf. virus (26)
FC	> 0.05	> 0.05	< 0.001	0.001	0.007	> 0.05	> 0.05	< 0.001
E. coli		> 0.05	0.010	0.019	0.040	> 0.05	> 0.05	< 0.001
Enterococci			> 0.05	> 0.05	> 0.05	> 0.05	> 0.05	0.006
SOMPH				> 0.05	> 0.05	> 0.05	> 0.05	> 0.05
F+PH					> 0.05	> 0.05	> 0.05	> 0.05
BSPH						> 0.05	> 0.05	> 0.05
Adenovirus							> 0.05	> 0.05
Norovirus								> 0.05

Values in parentheses represent number of observations used for ANOVA, statistically significant values are bolded. FC (fecal coliforms), SOMPH (somatic coliphages), F+PH (F+ coliphages), BSPH (Bacteroides spp. phages).

Overall, the greatest removal was achieved for culturable FIB, followed by molecular signal for pathogenic viruses, bacteriophages and finally infectious human enteric viruses (Figure 2). Specifically, reported and/or calculated log₁₀ reductions (± standard deviation) for FIB were as follows: 2.96 ± 2.07, 2.38± 1.26 and 2.22 ± 1.61 for fecal coliforms, E. coli and enterococci, respectively (Figure 2). The average removal of q(RT)-PCR signal of Adenoviruses and Noroviruses was approximately one log₁₀ lower compared to culture-based FIB and measured 1.62 ± 0.93 and 1.60 ± 0.85, respectively (Figure 2). Bacteriophage removal was similar to that for human viruses and averaged 1.46 ±1.24, 1.46 ± 1.18 and 1.29 ± 1.33 for somatic and F+ coliphages and Bacteroides spp. phages, respectively (Figure 2). Infectious human enteric virus removal was the lowest, averaging 0.90 ± 1.01 log₁₀ reduction (Figure 2).

Figure 2.

Reported and/or calculated log₁₀ reduction of FIB, bacteriophages and viral pathogens through wastewater treatment processes. Reduction data per organism type are pooled irrespective of the particular unit of operation (e.g. primary treatment, secondary treatment, disinfection). Box is delimited by 25^th and 75^th percentiles, solid line within the box represents median and dashed line represents average. Whiskers are 10^th and 90^th percentile values. Values outside of the range are depicted as filled dots. Number in parenthesis indicate number of studies* used to derive the data.

*References: (Bonadonna et al. 1993; Chauret et al. 1999; Baggi et al. 2001; Bourrouet et al. 2001; Rose et al. 2001; Tanji et al. 2002; Lucena et al. 2004; Harwood et al. 2005; Haramoto et al. 2006; Mandilara et al. 2006; Ottoson et al. 2006; Zhang and Farahbakhsh 2007; Carducci et al. 2008; Costan-Longares et al. 2008; Gomila et al. 2008; Petrinca et al. 2009; Aw and Gin 2010; Boczek et al. 2010; Fu et al. 2010; Ebdon et al. 2012; Flannery et al. 2012; Francy et al. 2012; Muniesa et al. 2012a; Hata et al. 2013; Pradhan et al. 2013; Kitajima et al. 2014; Ulbricht et al. 2014; Dias et al. 2015; Haramoto et al. 2015; Montazeri et al. 2015; Yahya et al. 2015; Mayer et al. 2016).

Comparison of compiled data (Table 1) indicated that fecal coliform and E. coli log₁₀ reductions were significantly higher compared to those of bacteriophages and infectious enteric viruses (P value range 0.019–<0.001), but there was no significant difference when compared to human viral gene targets (i.e. Adenoviruses and Noroviruses) (P>0.05). Enterococci removal was significantly greater compared to infectious enteric viruses (P=0.006), but there were no other statistically significant differences (P>0.05). Furthermore, there were no statistically significant differences when comparing removals of bacteriophages to those of infectious human enteric viruses, including Adenoviruses and Noroviruses (P>0.05). This finding suggests that bacteriophages may be better suited to act as surrogates for human viral pathogen removal through wastewater treatment processes compared to culturable FIB measurements.

Occurrence in the environmental waters

Freshwater

In general, freshwater concentrations of FIB were observed at higher levels than both bacteriophages and human enteric viruses. Results from a literature analysis showed mean concentrations of fecal coliforms, E. coli and enterococci atlog₁₀ 3.46 ± 1.41, log₁₀ 3.59 ±1.70 and log₁₀ 3.00 ± 1.47 CFU/100 mL, respectively (Figure 3). Among bacteriophages, somatic coliphages were detected at the highest concentrations ranging from 0.95 log₁₀ PFU/100 mL to upwards of 5.00 log₁₀ PFU/100 mL in more heavily impacted freshwaters with mean levels of log₁₀ 3.18 ±1.35PFU/100 mL typically observed (Figure 3). F+ coliphage levels in surface freshwaters averaged log₁₀ 2.00 ±1.29 PFU/100 mL mean levels, approximately log₁₀ 1–1.5 lower than somatic coliphages (Figure 3). While detectable levels of Bacteroides phage are found in environmental waters, they were less prevalent than somatic coliphages. In freshwaters, levels of Bacteroides phages (all types combined) were detected at mean concentrations of log₁₀ 2.00±1.12 PFU/100 mL, levels similar to that of F+ coliphages, but an order of magnitude lower than mean levels of somatic coliphages (Figure 3). Of the three Bacteroides phages, GB124 and HSP40 were detected at higher mean levels (log₁₀ 2.31±1.26 and log₁₀ 2.29±1.14 log₁₀ PFU/100 mL, respectively) compared to RYC2056 (log₁₀ 0.9±0.7 mean PFU/100 mL). Phages infecting E. faecalis were detected at the lowest levels, averaging log₁₀ 1.63±1.02PFU/100 mL, approximately log₁₀ .5 PFU/100 mL lower than Bacteroides phages and F+ coliphages, and ~log₁₀ 1.5 PFU/100 mL lower than that of somatic coliphages. Infectious enteric viruses were detected at lower concentrations than both FIB and bacteriophage, with mean levels of log₁₀ −1.27 ± 0.95 PFU/100 mL reported (Figure 3). Levels of Adenovirus (log₁₀ 1.67 ± 2.11 genomic copies/100 mL), Norovirus GI and GII (log₁₀ 0.63 ± 0.92 genomic copies/100 mL) and Rotavirus (log₁₀ 0.57 ±1.30 genomic copies/100 mL) determined by q(RT)-PCR in freshwater were higher than infectious enteric virus concentrations (Figure 3).

Figure 3.

Mean concentrations and 95% confidence intervals of FIB and bacteriophages (expressed as CFU, FU or MPN), as well as viral pathogens (expressed as MPN for infectious viruses or genomic copies for all others) in freshwater (empty symbols) and marine waters (filled symbols). The size of the symbol is proportionate to the number of studies* (given in parentheses) used to derive the data.

*References: (Tartera et al. 1989; Cornax 1991; Lucena 1996; Araujo et al. 1997; Muniesa et al. 1999; Contreras-Coll et al. 2002; Duran et al. 2002; Skraber et al. 2002; Hot et al. 2003; Lucena et al. 2003; Moce-Llivina et al. 2003; Choi and Jiang 2005; Lodder and de Roda Husman 2005; Montemayor et al. 2005; Ebdon et al. 2007; Ibarluzea et al. 2007; Boehm et al. 2009b; Lodder et al. 2010; Vijayavel et al. 2010; Gomez-Donate et al. 2011; Lee et al. 2011; Nnane et al. 2011; Viau et al. 2011; Santiago- Rodriguez et al. 2012; Wyer et al. 2012; Love et al. 2014; Rezaeinejad et al. 2014; Vijayavel 2014; Vergara et al. 2015; Santiago-Rodriguez 2016).

Marine Water

In marine waters, mean concentrations of fecal coliforms, E. coli and enterococci were measured at log₁₀ 2.88 ± 2.95, log₁₀ 1.87 ± 0.58and log₁₀ 2.03 ± 1.33 CFU/100 mL, respectively (Figure 3). Overall, FIB concentrations were one to two orders of magnitude lower compared to freshwaters samples with the largest difference observed for E. coli (Figure 3). Mean somatic coliphages in marine waters (log₁₀ 1.66 ± 1.73) were up to log₁₀ 1.5 PFU/100 mL lower than the levels in freshwater, and ranged from no detection to log₁₀ 6.28 PFU/100 mL (Figure 3). Recorded concentrations of F+ coliphages in marine waters were lower than those of somatic, averaging log₁₀ 0.89±2.23 PFU/100 mL (Figure 3), an order of magnitude below levels observed in the freshwater. Bacteroides spp. phage (all types combined) concentrations in marine waters ranged from no detection to log₁₀ 4.26 PFU/100 mL, with mean concentrations of log₁₀ 1.52 ± 2.37 PFU/100 mL, log₁₀ 0.5 PFU/100 mL lower compared to freshwater (Figure 3). There is currently insufficient data detailing the concentrations of Enterococcus phages or human enteric viruses in marine water to be included in these analyses.

Factors influencing fate and transport of bacteriophages

Through various mechanisms such as combined sewer overflows (CSOs), WWTP discharges, failing septic systems, or fecal deposits, bacteriophages can enter environmental waters where they are subject to various selective pressures and environmental insults that govern their fate and transport. Here we briefly examine some biotic and abiotic factors influencing survival of bacteriophages in the environment and provide a comparison with FIB and human enteric viruses when possible. Scarcity of the available data necessitated pooling the information together irrespective of the study design (e.g. laboratory, bench scale or field study) or the coliphage origin (e.g. prototypes such as MS2 and ΦX174 as opposed to indigenous organisms).

Depending on factors such as viral surface charge, pH of the water and levels of suspended solids in water column, bacteriophages and enteric viruses are often attached to particulate matter in aquatic environments. However, the processes of viral dispersion, transport and subsequent attenuation are poorly defined (Ferguson 2003). Attachment to particles is particularly important when considering the movement since it can partition the organisms and therefore prevent them from reaching the surface or ground waters. Attachment of coliphages to particles can be highly variable (ranging from 20% to 60%) and is influenced by environmental factors, as well as heterogeneity of different bacteriophage groups (Characklis et al. 2005). This partitioning behavior seems to be somewhat similar to that of infectious enteric viruses whereby 72 to 78% of samples containing infectious virions are associated with particles as opposed to 14 to 50% found in the overlaying water column (Rao et al. 1984). Studies of coliphage transport indicate that the attachment to particles is the most important process in phage removal, while detachment rates are typically 100 to 1000 times lower (Hijnen et al. 2005), although this process can be considerably influenced by the oxygen content with greater adsorption observed in more oxic environments (Frohnert et al. 2014). By comparison, FIB attachment to particles has been reported to be somewhat more constant (20–55%) (Characklis et al. 2005), a finding that could be attributed to a more uniform isoelectric point of bacteria (Harden and Harris 1953; Jewett et al. 1995; Stevik et al. 1999).

Bacteriophage transport is also dependent on the type of soil/sediment as well as velocity of the surrounding water (Cho et al. 2016). For example, coarse-grained, heterogeneous, gravel aquifers with high velocities have low filtration capacity and to achieve estimated 7 log₁₀ removal of MS2 bacteriophage it would require a setback distance of 3.9 km, but would require only ~129 meters in sandy fine gravel (Pang et al. 2005). The possibility of a “shielding effect” conferred by particle association (Templeton et al. 2005) combined with relatively high concentrations of coliphages (Chung 1993; Paul et al. 1993; Karim et al. 2004; Skraber et al. 2009; Yamahara et al. 2012) and enteric viruses (Smith et al. 1978; LaBelle and Gerba 1979; Rao et al. 1984) typically recorded in sediments and soils (as compared to the overlaying water columns), suggests that they can act as virus reservoirs. In comparison, FIB appear to be similarly influenced by the soil type but less affected by the velocity which is not surprising consider the straining effect of porous media (Ferguson 2003; Hijnen et al. 2005). Similar to coliphages, sediments can also act as reservoirs of FIB (Yamahara et al. 2012). Human activities, such as boating and swimming, along with changes in water currents and activities of wildlife, can resuspended and subsequently physically desorb viruses and bacteria attached to sediment particles therefore potentially increasing the viral loads in the water column (Ferguson 2003).

The effect of temperature on survival of bacteriophages is one of the most studied environmental factors. Numerous experiments demonstrated extended survival at lower temperatures in variety of matrices including sediments/soils (Hurst et al. 1980; Chung 1993; Gantzer et al. 2001; Ottosson and Stenstrom 2003), freshwaters (Long and Sobsey 2004; Skraber et al. 2004b; Yang and Griffiths 2013; Ravva and Sarreal 2016), marine water (Chung 1993), wastewater (Moce-Llivina et al. 2003; McMinn et al. 2014), groundwater (Yates et al. 1985; Gordon and Toze 2003) and tap water (Allwood et al. 2003). In general FIB decay is accelerated compared to bacteriophages and enteric viruses at all of the temperatures examined, while bacteriophages typically survived longer than infectious enteric viruses (Yates et al. 1985; Chung 1993; Gantzer et al. 2001; Allwood et al. 2003; Gordon and Toze 2003; Moce-Llivina et al. 2003; Skraber et al. 2004b; Charles et al. 2009). Somatic coliphages typically persisted longer than the F+ group (Gantzer et al. 2001; Moce-Llivina et al. 2003; McMinn et al. 2014), with different genotypes of the latter exhibiting differential survival characteristics(Long and Sobsey 2004; Muniesa et al. 2009; Yang and Griffiths 2013; Ravva and Sarreal 2016). Survival of Bacteroides spp. phages varied, with some strains behaving more similarly to F+ (Duran et al. 2002; McMinn et al. 2014), while others resembled somatic subgroup more closely (Duran et al. 2002; Moce-Llivina et al. 2003).

Another important factor for bacteriophage survival is sunlight and associated UV radiation. Irrespective of the source of radiation (simulated or ambient sunlight), reported decay of FIB, bacteriophages and enteric viruses were always lower in dark treatments as opposed to sunlight exposed ones (Sinton et al. 1999; Sinton et al. 2002; Noble et al. 2004; Silverman et al. 2013; United States Environmental Protection Agency 2015; Sun et al. 2016). Comparable to the effect of temperature, FIB decay was always greater compared to that of bacteriophages and viruses, but the magnitude of the effect on different microorganisms seems to be at least somewhat dependent on the water type, as differential decay metrics are reported for freshwater compared to marine waters (Sinton et al. 1999; Sinton et al. 2002; Silverman et al. 2013; United States Environmental Protection Agency 2015). Sunlight wavelength (UVA, UVB and visible spectrum) (Sinton et al. 1999; Sinton et al. 2002; Fisher et al. 2011; Silverman et al. 2013; United States Environmental Protection Agency 2015; Sun et al. 2016) as well as the presence of natural organic matter (Silverman et al. 2013; United States Environmental Protection Agency 2015; Sun et al. 2016) are also reported to influence the magnitude and type of bacteriophage inactivation (exogenous, direct and indirect endogenous). Lastly, it is important to note that decay studies characterizing seeded coliphage laboratory strains exhibit different transport and survival characteristics (Brion et al. 2002; Long and Sobsey 2004; Ravva and Sarreal 2016) compared to indigenous organisms. As a result, generalizations combining coliphage survival from spiking experiments and environmental isolates is not prudent.

Summary

Bacteriophages are alternative fecal indicator organisms that are better suited than FIB to act as surrogates for enteric viral pathogens presence and removal in built and natural environments. Our review suggests that removal of bacteriophages through wastewater treatment plants was more similar to that of infectious viral pathogens than FIB to infectious viral pathogens, suggesting the potential advantage of their use as alternative viral indicators in this arena. While bacteriophage densities in fresh and marine waters are lower compared to FIB, but higher than viral pathogens, the accurate assessment of their concentrations is confounded by the lack of recovery data for these matrices. Additionally, there is evidence that bacteriophage fate and transport in the ambient waters may resemble that of viral pathogens more closely than FIB and therefore bacteriophages may be promising surrogates under some environmental conditions.