Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters Tasha M. Santiago-Rodrı´guez a, Catalina Da´vila a,*, Joel Gonza´lez b, Natasha Bonilla a, Patricia Marcos c, Miguel Urdaneta a, Manuela Cadete d, Sı´lvia Monteiro d, Ricardo Santos d, Jorge Santo Domingo b, Gary A. Toranzos a a

Environmental Microbiology Laboratory, Department of Biology, University of Puerto Rico, Rico, San Juan 00979, Puerto Rico US Environmental Protection Agency, Cincinnati, OH, USA c Biological Sciences Department, General Studies, University of Puerto Rico, San Juan, Puerto Rico d Instituto Superior Tecnico, Laboratorio de Analises, Lisbon, Portugal b

article info

abstract

Article history:

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and

Received 28 April 2010

have been recently isolated from environmental water samples. Although enterophages

Received in revised form

have not been conclusively linked to human fecal pollution, we are currently charac-

2 July 2010

terizing enterophages to propose them as viral indicators and possible surrogates of

Accepted 20 July 2010

enteric viruses in recreational waters. Little is known about the morphological or genetic

Available online 7 August 2010

diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped

Keywords:

by their ability to replicate at different temperatures, and if different groups are present

Enterophages

in the feces of different animals. As one of the main objectives is to determine if these

Enterococci

phages can be used as indicators of the presence of enteric viruses, the survival rate

Fecal pollution

under different conditions was also determined as was their prevalence in sewage and

Indicators

a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T90 values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution. ª 2010 Elsevier Ltd. All rights reserved.

* Corresponding author. E-mail address: [email protected] (C. Da´vila). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.07.078

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

1.

Introduction

Contamination of water sources by sewage is a health-related risk because of the possible presence of pathogenic microorganisms such as Salmonella, Campylobacter, enteric viruses as well as pathogenic protozoa (Hurst, 1996; Moe, 1996; Toranzos et al., 1996; Fong and Lipp, 2005). Thermotolerant coliforms (Wolf, 1972; Ashbolt et al., 2001), Bacteroides fragilis phages (Jofre et al., 1995; Gantzer et al., 1998) and somatic and F(male)specific coliphages (Stetler, 1984; Grabow and Coubrough, 1986; Hernandez-Delgado et al., 1991; Gantzer et al., 2001; Allwood et al., 2003) are used and have been proposed to infer the presence of these pathogens in water sources. However, these microorganisms fail to fulfill the criteria of an ideal microbial indicator. For instance, the prevalence and survival of thermotolerant coliforms is short and cannot be correlated with that of pathogens (Toranzos et al., 1996; Bonilla et al., 2010), human pathogens are not always accompanied by enterococci and vice versa (Weisberg et al., 1996; Baele et al., 2002; Cox and Gilmore, 2007) and B. fragilis phages are found only in some geographical areas (Weisberg et al., 1996). The survival of somatic and Fþ RNA and Fþ DNA coliphages is approximately 20e100 days at 20  C in fresh water, depending on the group, which cannot be correlated with the survival of enteric viruses (Allwood et al., 2003; Long and Sobsey, 2004). Yet, other studies have found that Fþ RNA coliphages could be active as long as 3 days and somatic coliphages as long as 7 days (Moce-Llivina et al., 2005). Even though serotypes II and III of Fþ RNA coliphages have been correlated with human fecal contamination (Furuse et al., 1981; Scott et al., 2002; Long and Sobsey, 2004), their presence in cow, pig, horse and bird feces makes them unreliable markers of specifically human fecal pollution (Tavakoli et al., 2002; Cole et al., 2003; Kelly and Sanderson, 1960). Also, currently used indicators are very susceptible to standard chlorination treatments (Tavakoli et al., 2002), while enteric viruses like Coxsackie virus, Rotavirus and Norwalk viruses are susceptible to higher chlorine concentrations and have been found in treated sewage (Kelly and Sanderson, 1960; Keswick et al., 1984; Keswick et al., 1985; Payment and Franco, 1993). Because the presence of enteric viruses in water sources is of great concern due to their resistance to removal treatments (Keswick et al., 1985), new and reliable indicators of human fecal pollution and the possible presence of these pathogens are still needed. Therefore, we are proposing bacteriophages that specifically infect Enterococcus faecalis, which we call enterophages, as viral indicators of human fecal contamination. Enterophages are a new group of phages that have been recently isolated from recreational waters. Most isolates are tailed-phages, with icosahedral capsids of 80 nm in diameter and tails of 200 nm in length (Bonilla et al., 2010), while others have smaller capsids (12 nm) and shorter tails (60 nm) (this study), which accordingly could belong to the Siphoviridae family (Bonilla et al., 2010). These isolates differ in size from other previously characterized E. faecalis-infecting phages, which have bigger capsids and tails (93 nm and 204 nm, respectively) (Bachrach et al., 2003); other isolates do not possess tails and have sphere-shaped, spiked structures of

4717

approximately 70 nm (Uchiyama et al., 2007). We have previously developed the methods for their detection in environmental samples and results showed that these are promising markers of human fecal pollution. The survival of enterophages in marine recreational waters was seen to be approximately 7e10 days (Bonilla et al., 2010), similar to that of enteric viruses under similar environmental conditions (Ward et al., 1986; Bonilla et al., 2010) and unlike bacterial fecal indicators, enterophages have not been detected in pristine ecosystems (Bonilla et al., 2010). Even though results show that enterophages possess some of the characteristics of an ideal viral indicator of water contamination by human feces, further characterization is still needed. It still remains unknown how many groups of enterophages can be present in water contaminated with feces, though it has been suggested that at least three groups exist (Bonilla et al., 2010). Previous studies have found that different groups of coliphages exist and can be grouped according to their ability to replicate at specific temperatures (Seeley and Primrose, 1980). Consequently, the aims of the present study are to determine if enterophages are present in animal and human fecal samples, to characterize enterophages by means of their morphology, composition of their genetic material and their ability to replicate at some temperatures and not others; to determine the prevalence of enterophages in a fresh water gradient in the central region of Puerto Rico as well as to determine if there are temporal variations in sewage in Puerto Rico and Portugal, and to determine their survival in fresh and marine waters. Because coliphages have been used and proposed as water quality controls of fecal contamination, their detection in both animal and human feces, as well as the prevalence and survival studies were done in parallel with that of enterophages as a means of comparison. The survival of enterophages in sand was also performed because of the possible introduction of pathogens by feces. Most bacteria are not reliable indicators of fecal contamination in beach sand because these are part of the environmental microbiota (Khiyama and Makemson, 1973).

2.

Materials and methods

2.1. feces

Detection of enterophages in animal and human

Cattle was selected as a study model to detect enterophages because E. faecalis is the most commonly enterococci found in their feces (Devriese et al., 1992). Eight grams of cattle feces from 15 different animals were collected from 4 different farms and stored at 20  C until processed. To detect enterophages, 1 g of each sample, 0.1 mL of Tween-20 (Golomidova et al., 2007), were added to 50 mL of sterile distilled water and processed using the single layer method. The suspension was added to an equal volume of liquefied regular strength (1) Tryptic Soy Broth (TSB) (Difco) þ agar (0.75% w/v) (Difco) which had CaCl2$2H20 (Fisher Scientific Co. NJ, USA) and NaN3 (MCB, OH, USA) (final concentration of 2.6 mg/mL and 0.4 mg/mL, respectively). Additionally, 5 mL of an overnight culture of E. faecalis grown in Azide Dextrose Broth (Difco) were added to the mixture. This method was similarly conducted for the

4718

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

detection of coliphages. Briefly, the suspension was added to molten 2-strenght TSB and agar (1.5% w/v). One mL of a fresh culture of Escherichia coli in TSB was used for their detection. To test the presence of different groups of coliphages and enterophages (according to their ability to replicate at different temperatures), the mixtures were poured into sterile Petri dishes and incubated at 22, 37 or 41  C for 24 h (Bonilla et al., 2010). To detect enterophages and coliphages in humans, fresh feces from 5 healthy individuals were processed as follows: 0.1e0.5 g of fresh feces were suspended in 3 mL of a sterile 0.85% NaCl solution. 0.1 and 1 mL of the suspension were separately processed using the double layer method. Briefly, the volumes were added to 4 mL of molten 1 TSB þ agar (final 0.75% w/v), which had CaCl2 and NaN3 as described in section 2.1. Three mL of an overnight culture of E. faecalis were added to the mixture and poured into petri dishes containing 20 mL of 1 TSB þ agar (final 1.5% w/v). For the detection of coliphages, 1 mL of an overnight culture of E. coli was added to the mixture. Plates were incubated at 37  C for 24 h.

2.2.

Enterophage isolation and purification

To isolate enterophages for further characterization, a 50 mL volume sample of raw domestic sewage was processed as described in section 2.1 (Bonilla et al., 2010). The solution was mixed, poured into sterile Petri dishes and plates were incubated at different temperatures, namely 22, 37, 41 and 45  C for 24 h. Individual viral plaques detected at each of the incubation temperatures were isolated. Different plaque size and translucency were used as criteria for the isolation. Plaques were plucked using sterile Pasteur pipettes and the plug was placed in a sterile Eppendorf tube containing 500 mL of sterile PBS. The plugs were dislodged and emulsified using the same pipette. The tubes were centrifuged at 14,000 rpm for 10 min at 10  C to remove cellular debris and agar and the supernatant transferred into a sterile tube. The isolates were then subcultured using 100 mL of the supernatant and processed using the double layer method as described in section 2.1. Then, for those plates showing complete viral lysis, 5 mL of sterile phosphate buffer saline (PBS) was added and slowly agitated rotationally for 20 min. The top agar was transferred to a sterile centrifuge tube, broken down and then centrifuged at 14,000 rpm for 10 min at 10  C. The supernatant containing the viruses was kept in a sterile tube at 4e7  C for further use. To determine if the isolates were capable of replicating at other temperatures, an E. faecalis inoculum was spread throughout a Petri dish containing 1 TSB, 0.75% agar, CaCl2 and NaN3 using a sterile swab. An aliquot of 10 mL of the purified supernatant was placed on top of the Petri dishes and incubated at 22, 37, 41 and 45  C for 24 h. Further titration of each isolate was done using the double layer method as described above.

2.3.

Morphological characterization of enterophages

Prior to the examination of the morphology of the enterophage isolates, hydroextraction was used to concentrate the viruses; briefly, a dialysis tube (12,000e14,000 MW cutoff, Spectrapor, Los Angeles, CA) was loaded with 5 mL of the enterophage isolate, covered with crystalline polyethylene

glycol (PEG, Mol.wt. 8000, Sigma Chem. Co. MO; APHA, 1989), clamped and placed at 4e7  C overnight. The dialysis tube was washed with 100 mL of sterile 0.85% NaCl. The resulting solution was transferred into a sterile Eppendorf tube and kept at 4  C until analysis. An aliquot of the final solution was placed on carbon Type-B 200 mesh copper grids or ultra thin carbon film/holey carbon 400 mesh copper grids and stained with uranyl acetate 2%, pH 4.5. All specimens were examined using a Karl Zeiss Leo 922 energy filtered transmission electron microscope operated at 200 KV. At least 5 phage particles of each type were observed (Bonilla et al., 2010).

2.4.

Nucleic acid analyses

Extraction of enterophages nucleic acids was conducted using standard techniques of proteinase K and phenol:chloroform treatments, followed by precipitation with ethanol (Sambrook et al., 1989). To determine if the enterophage isolates genetic material was DNA or RNA, aliquots of the isolated nucleic acids were treated with either 1 U/ml DNase (Promega, Madison, WI) (Huang et al., 1996) or 10 mg/ml RNase (SigmaeAldrich, Co. St. Louis, MO, USA). Agarose gel electrophoresis was performed as previously described (Sambrook et al., 1989), using 0.7% of agarose (SigmaeAldrich, Co. St. Louis, MO, USA) gels in TAE buffer. Bands were visualized after staining with an ethidium bromide solution (final concentration of 0.5 mg/ml).

2.5.

Prevalence in raw sewage

To determine the prevalence of enterophages in raw and treated domestic sewage from Puerto Rico and Portugal, samples were collected monthly for 6 months and processed using the single layer method as described in section 2.1 (Bonilla et al., 2010). For the detection of coliphages, serial dilutions were done and processed using the double layer method as described in section 2.1. Plates were incubated at 37  C for 24 h.

2.6. Detection of enterophages and other viral and bacterial indicators in a large watershed Ten sampling sites subjected to different environmental conditions in the Rio Grande de Arecibo watershed, localized in the central region of Puerto Rico, were selected to determine the prevalence of enterophages in a natural setting (Fig. 1). One liter samples from each point were collected in sterile plastic bottles every week for 2.5 months and kept at 8  C until processing. Samples were analyzed for thermotolerant coliforms, enterococci, coliphages and enterophages (USEPA, 2002; Bonilla et al., 2010). For bacteria enumeration, 100 mL of water per sample site were filtered using 13 mm polycarbonate membrane filters (GE Water & Process Technologies, 0.45 mm). This was done separately for the enumeration of both bacterial indicators. For the enumeration of thermotolerant coliforms, filters were placed on Difco m-FC agar and incubated at 45  C for 24 h. Enumeration of enterococci was done by placing the membrane filter on Difco m-Enterococcus agar and incubated at 37  C for 48 h. Coliphage and enterophage were enumerated by processing 100 mL of the water samples using the single layer method as described in section 2.1 (Bonilla et al., 2010). The solution was

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

4719

Fig. 1 e Shows study site. Sample points are ordered according to their position in the watershed, localized in the central region of Puerto Rico. Rio Grande de Arecibo watershed localized in the central region of Puerto Rico. Sample points in this study are ordered according to their position in the watershed (See legend).

poured into Petri dishes and incubated at 22, 37, 41 or 45  C for 24 h to test for the possible presence of different groups of enterophages across the watershed.

2.7. Survival of enterophages and coliphages in waters and sand To determine the survival of enterophages and coliphages in fresh and marine waters as well as in beach sand, 2 L of fresh

or marine water samples and 2 kg of sand were collected in sterile plastic bottles and kept at 8  C till used. At the laboratory, the water and sand samples were placed in a sterile covered glass beaker. In order to simulate the die-off rate of these bacteriophages under natural conditions (Noble et al., 2003; Duran et al., 2001), raw domestic sewage with a coliphage concentration of 104/100 mL was added to the samples; the same concentration of an enterophage isolate from a laboratory stock was also added to the samples. The samples

4720

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

were kept in the dark at 22  C for up to 12 days. Every two days, 50 mL aliquots were separately processed for the detection of enterophages and coliphages using the single layer method as described in section 2.1. Similarly, 50 g of sand and 0.1 mL of Tween-20 were added to 50 mL of sterile distilled water and vigorously shake. The suspensions were processed as previously described in section 2.1 (Bonilla et al., 2010). In order to make comparisons, decay constants (kd) from both enterophages and coliphages in fresh and marine waters and sand were determined using the slopes of linear regressions made on the semilog plots (PFU versus time). Decay rates (percent/2 days) were calculated by multiplying the decay constants by 100. The time to reach a 90% reduction in PFU densities (T90) was also determined by dividing ln (0.1)/kd (Noble and Fuhrman, 1996; Sinton et al., 1994, 2007; Noble et al., 2003).

3.

Results and discussion

3.1. feces

Enterophages and coliphages in animal and human

Coliphages have been used as viral indicators of fecal pollution, but their host specificity includes cows, swine, humans, etc. In addition, those groups found in humans can also be found in animal feces (Cole et al., 2003). Similarly to these studies, we found coliphages in 12 of the 15 cattle feces samples at an incubation temperature of 22  C, but not at other incubation temperatures. Their concentrations ranged from 8 PFU to 326 PFU/g (data not shown). On the other hand, enterophages were not detected in any of the cattle feces. Even though enterophages were not detected, additional fecal samples from different animal species must be processed to confirm enterophages specificity. Coliphages were detected in 3 out of the 5 human fecal samples (approximately 80 PFU/g;

Fig. 2 e Transmission Electron Microscopy image (TEM) of an enterophage isolate in this study. The image shows that the isolate possesses an icosahedral head of 12 nm and a 60 nm non-contractile tail (Bar [ 20 nm).

Fig. 3 e The genetic material of several enterophage isolates in a 0.7% agarose gel. The molecular weight marker is shown in lane M (Lambda DNA HindIII digest, SigmaeAldrich Co. St. Louis, MO, USA). Lanes 1 and 4 contain untreated nucleic acids from two different isolates. Lanes 2 and 5 contain DNase treated samples and lanes 3 and 6 contain RNase-treated samples.

data not shown), but enterophages were not detected. These results could be due to the low concentrations of enterophages found in human feces. Also, other incubation temperatures must be tested for their detection in fecal samples (namely 22 and/or 41  C). Regarding the method used, the single layer method could be more appropriate for their detection in human feces. These results are similar to those found in other studies which showed that bacteriophages may be present in human feces at concentrations ranging from 0 to 105 CFU/g (Furuse et al., 1983; Cornax et al., 1994; Gantzer et al., 2002; Breitbart et al., 2003). These studies have found that many of the bacteriophages in the human intestine could be those infecting gram-positive bacteria, as in the case of enterophages (Breitbart et al., 2003). The absence of enterophages in cattle feces and other animals (Bonilla et al., 2010) is encouraging, since it suggests that these viruses are restricted to human hosts and are present in low numbers in the colon.

Fig. 4 e Enterophage concentration/mL of four different enterophage isolates among four different temperatures. Group 1 and 2 represent phages isolated from 22  C, group 3 represents an isolate from 37  C and group 4 represents the isolate from 41  C. Results represent the geometric mean of three replicas and standard deviations are represented by error bars.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

Table 1 e Prevalence of enterophages in raw and treated sewage at different domestic wastewater treatment plants in Puerto Rico (PR) and Portugal (Port). All data represent the average concentration and standard deviation of viral plaques over a six-month period (n [ 6). Water Treatment Plant PR-A PR-B PR-C PR-D Port-A Port-B Port-C Port-D Port-E Port-F Port-G Port-H Port-I Port-J

Enterophages in Enterophages in influent (PFU/100 mL) effluent (PFU/100 mL) 82 60 115 53 17 634 695 22 575 212 405 173 774 487

             

51.5 46.1 140.1 53.5 3.2 15.7 21.6 2.8 22.9 7.0 6.6 10.9 11.2 6.5

56  14  18  6 1 253  3 2 5 12  11  21  3 4

55.6 14.5 1.2 10.7 0.9 4.6 0.9 0.6 1.3 1.3 1.4 1.7 0.9 0.9

3.2. Differences in enterophage isolates according to morphology, genetic material and ability to replicate at different temperatures Both the morphology and genetic material of the enterophage isolates in this study correspond to those viruses belonging to the Siphoviridae family. The morphology of the enterophage isolates described here indicates that these are tailed-phages, with icosahedral capsids of 12 nm in diameter and non-

4721

contractile tails of 60 nm long (Fig. 2). This morphology is different from other virions we have described recently (Bonilla et al., 2010), which showed a bigger capsid and a longer tail, similar to the classical Bradley’s basic morphology Group B belonging to the Siphoviridae family (Fourth report of the International Committee on Taxonomy of Viruses, 1982). In terms of the genetic material of enterophages, high molecular weight bands were observed in the Rnase-treated samples. Enterophage nucleic acid molecular weight appears to be more than 23 kb and be in a super coiled conformation. Fragments in lanes 2 and 5 were degradated by DNase, showing that the genetic material of the enterophage isolates described here is composed of DNA (Fig. 3). However, it still remains the possibility that the morphology and genomes of other isolates could be different from those described. It also remains unknown if the isolates in this study are double stranded or single stranded DNA viruses. Enterophages isolated at 22  C were capable of replicating at 22, 37, 41 and 45  C. However, those enterophages that were isolated at 37 and 41  C replicated at 22, 37 and 41  C, but not at 45  C (Fig. 4). Though further characterization of enterophages is still needed, these results suggest that at least two groups of enterophages exist. Because enterophages can be isolated at environmental temperatures, they can be detected in places without the facility of an incubator by incubating the plates at ambient temperatures.

3.3. Prevalence of enterophages in sewage and in a large watershed in Puerto Rico Although enterophages were isolated at lower concentrations than coliphages, they were detected in raw and treated domestic sewage in this study (Table 1). Interestingly, even

Fig. 5 e Concentrations of enterophages (A), coliphages (B), enterococci (C) and thermotolerant coliforms (D) at ten different fresh water sample points with different impacts. Sample points are numbered according to their position in the watershed: (1) Garza lake, (2) Vaca river, (3) Before WTP Adjuntas, (4) After WTP Adjuntas, (5) Rio Grande de Arecibo, (6) Before WTP Utuado, (7) After WTP Utuado, (8) Mouth of Rio Grande de Arecibo, (9) Criminales river and (10) Caguana river at Jayuya. Numbers represent the geometric mean of a 2.5 month sampling period. Standard deviation is represented by error bars.

4722

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

Fig. 6 e Survival of enterophages (A) and coliphages (B) in fresh water. Numbers represent the arithmetic mean of three replicas. Standard deviations are represented by error bars.

though the same host strain was used to detect enterophages in both Portugal (Port) and Puerto Rico (PR) treatment plants, they were found about 1 log10 higher in raw domestic sewage from Portugal. Prevalence studies in Portugal indicate that these viruses are not restricted to particular geographical areas and could be tested as viral indicators in different water types. Enterophages were also detected in treated sewage in Puerto Rico, as well as coliphages (data not shown). Primary treatment is performed on raw sewage from domestic wastewater treatment plants PR-A, PR-B and PR-C, while raw sewage from PR-D receives tertiary treatment. In raw sewage receiving primary treatment, approximately 56e97% of enterophages were removed (Table 1), while a 91% removal was seen in sewage from wastewater treatment plant PR-D. Coliphages, on the other hand, were removed from a 95 to a 98% in PR-A, PR-B and PR-C, whereas more than 99% of coliphages were removed from raw sewage in PR-D (data not shown). A reduction of approximately 1e2 log10 of enterophages was seen in raw sewage from Portuguese treatment plants (Table 1). Prevalence results from Puerto Rican treatment plants suggest that enterophages may be more resistant to removal treatments than coliphages and could be potentially more useful as indicators of the presence of enteric viruses even in treated sewage. In the large watershed, coliphages were found at higher concentrations than enterophages in all sample points, but both bacteriophages were found at lower concentrations than thermotolerant coliforms and enterococci (Fig. 5). The high concentrations of bacterial indicators in the watershed suggest that these may also be from animal origin,

Fig. 7 e Survival of enterophages (A) and coliphages (B) in marine water. Numbers represent the average of three replicas. Standard deviations are represented by error bars.

consequently, using these to track the source of the contamination may not be reliable (Field and Samadpour, 2007; Bae and Wuertz, 2009). No fluctuations in enterococci and thermotolerant coliforms concentrations were found throughout the sampling period (Fig. 5C and D, respectively). Coliphages were detected at all incubation temperatures in most of the sample points. These results suggest that different groups of coliphages were introduced into the water sources, which may not necessarily be of human origin. Coliphage concentrations were found to fluctuate throughout the sampling period; however, fluctuations were not as prominent as those of enterophages (Data not shown). We are in the process of further characterizing the enterophage isolates molecularly, including the sequencing of the viral genomes, which may allow us to determine unique sequences to be used for the development of real-time methods for their detection in recreational and other water sources. Also, future epidemiological studies should be focused on determining the relationship between the prevalence of enterophages and enteric viruses in recreational waters and the symptoms associated with ingesting water testing positive for the presence of enterophages.

3.4.

Survival of enterophages in fresh waters

Enterophages were detected at 37 and 41  C at 4 h after the incubation of the Petri dishes, but not at 22  C (data not shown). This indicates that these temperatures can be used to

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

4723

fecal contamination in water sources. Future survival studies of enterophages in fresh waters will be done in order to determine the survival of enterophages and coliphages in different points across the watershed described here and those factors that could potentially affect their survival.

3.5. Survival of enterophages in marine recreational water and sand

Fig. 8 e Survival of enterophages (A) and coliphages (B) in sand. Numbers represent the average of three replicas. Standard deviations are represented by error bars.

detect enterophages in water samples in less time than bacterial indicators at similar temperatures. Enterophages have a survival similar to that of human enteric viruses in fresh waters (Fig. 6A) (Ward et al., 1986; Moce-Llivina et al., 2005) and are able of replicating at 22, 37 and 41  C. On the other hand, the survival of coliphages in this study was more than 12 days, which cannot be correlated with that of enteric viruses (Fig. 6B). Even though temperature does not affect the formation of viral plaques, it seems to affect the decay rates of enterophages. At 22 and 37  C, the decay rates were 33.7 and 38.5% every 2 days, respectively and at 41  C the inactivation rate was 35.9% every 2 days. T90 values also differed among the different temperatures. At 22 and 41  C, 90% of the initial inoculum was inactivated at approximately 6.8 and 6.6 days, respectively and 6.0 days at 37  C. Although it is not new that temperature does affect the decay rates of microorganisms (Noble et al., 2003; Long and Sobsey, 2004), our experiments on enterophages decay rates are part of their characterization as markers of human fecal pollution and further experiments are still needed. Temperature also affected the inactivation rates of coliphages. At 22  C, approximately 11.6% of the initial inoculum was inactivated every 2 days. At 37  C, coliphages had an inactivation rate of 9.9% every 2 days; but the highest inactivation rate was seen at 41  C (26.2% every 2 days). The time required to inactivate 90% of the initial coliphage concentration at 22  C was 20.0 days, 23.3 days at 37  C and 9.0 days at 41  C (Fig. 6B). The long die-off rate of coliphages indicates that some groups may not be used to track recent

As in the survival study of enterophages in fresh waters, plaques were detected at 37 and 41  C at 4 h after the incubation, but not at 22  C, which suggests that these temperatures are favorable to detect enterophages in short incubation periods. This could be due to diminished adsorption rates of enterophages at low temperatures possibly because of a reduced affinity between phages and bacterial surface receptors. Also, the adsorption rate is usually higher at the optimum temperature for the growth of the host bacteria, suggesting that actively functioning bacteria are required for a successful infection (Binetti et al., 2003). Nevertheless, after 11 days viral plaques were not detected at 4 h of incubation to any of the temperatures, but they were still detected after 24 h. This may indicate that the adsorption rates were reduced with time. The enterophage isolate used in this study has a survival time in both marine waters and sand of 11e13 days (Figs. 7A and 8A), which is less than coliphages (Figs. 7B and 8B). The decay rates were 27%, 29.3% and 30.7% every two days at 22, 37 and 41  C, respectively. The calculated T90 values for each temperature, were 8.5, 7.9 and 7.5 days correspondingly. In the study of enterophage survival in sand, the decay rates were 17, 20.6 and 21.4 and the calculated T90 values for each temperature, were 13.5, 11.2 and 10.8 respectively. Regarding coliphages, the decay rates in marine water were 5%, 4.5% and 7.7% every two days. As in the study of coliphages in fresh water, the higher decay rate occurred at 41  C. The decay rates for coliphages in sand were 3.6, 4.1 and 6.2 and the T90 values were 64, 56.2 and 37.1. Given that the isolate used in both survival studies (fresh water and marine water and sand) was the same, it is interesting that the observed survival time was higher in marine water and sand environments. Factors that could cause these longer survival periods include the high concentration of inorganic salts, due to the fact that phage adsorption is strongly dependent on salt concentrations (Moldovan et al., 2007). These results and previous survival experiments using coliphages have found that it would be impossible to correlate their survival with recent fecal contamination (HernandezDelgado and Toranzos, 1995). The survival of enterophages in fresh and marine waters could indicate the recent introduction of human feces into water sources. Molecular methods, like those currently tested on other microorganisms (Bae and Wuertz, 2009), could also be developed to determine the dieoff rate of enterophages in recreational waters. In addition, because the effect of sunlight has been extensively studied on bacteria and bacteriophages (Noble and Fuhrman, 1996; Noble et al., 2003; Bae and Wuertz, 2009; Sinton et al., 2007), the inactivation of enterophages by sunlight has to be tested in future studies.

4724

4.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

Conclusions

 The absence of enterophages in cattle feces could indicate their specificity to the human colon and could be potentially used to determine the presence of human enteric viruses in recreational waters.  Differences in morphology, genome and replication temperatures are factors that should be taken in consideration when characterizing a new viral indicator, as in the case of enterophages. These characteristics should be taken in consideration when choosing which enterophage isolates have the potential of being molecularly characterized for their detection in water sources.  Enterophages could be used as surrogates of enteric viruses in recreational waters due to their resistance to primary and tertiary treatments and similarity in die-off rates in fresh and marine waters.  Unlike proposed viral indicators, enterophages are not constrained to specific regions and could be used to infer human fecal pollution in different water sources.  Few indicators have been proposed to infer the introduction of pathogens into beach sand. The die-off rate of enterophages could also indicate recent fecal contamination, not only in water sources, but also in beach sand.

Acknowledgments We thank Carlos Toledo for reviewing the manuscript at its early and late stages, Marisol Rodriguez for collecting samples, Jean Frances Ruiz and Gwendolyn Argu¨ello for processing the samples.

references

Allwood, P.B., Malik, Y.S., Hedberg, C.W., Goyal, S.M., 2003. Survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in water: a comparative study. Appl. Environ. Microbiol. 69 (9), 5707e5710. APHA, 1989. Standard Methods for the Examination of Water and Wastewater. Seventeenth Edition. American Public Health Association, Washington DC. Ashbolt, N.J., Grabow, W.O.K., Snozzi, M., 2001. In: Fewtrell, L., Bartram, J. (Eds.), Indicators of Microbial Water Quality, in Water Quality: Guidelines, Standards and Health. IWA Publishing, London, U.K., pp. 289e316. Bachrach, G., Leizerovici-Zigmond, M., Zlotkin, A., Naor, R., Steinberg, D., 2003. Bacteriophage isolation from human saliva. Lett. Appl. Microbiol. 36 (1), 50e53. Bae, S., Wuertz, S., 2009. Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide. Water Res. 43, 4850e4859. Baele, M., Devriese, L.A., Butaye, P., Haesebrouck, F., 2002. Composition of enterococcal and streptococcal flora from pigeon intestines. J. Appl. Microbiol. 92 (2), 348e351. Binetti, A.G., Quiberoni, A., Reinheimer, J.A., 2003. Phage adsorption to Streptococcus thermophilus: influence of environmental factors and characterization of cell-receptors. Food Res. Int. 35, 73e83.

Bonilla, N., Santiago, T., Marcos, P., Urdaneta, M., Santo Domingo, J., Toranzos, G., 2010. Enterophages, a group of phages infecting Enterococcus faecalis and their potential as alternate indicators of human fecal contamination. Water Sci. Technol. 61, 293e300. Breitbart, M., Hewson, I., Felts, B., Mahaffy, J.M., Nulton, J., Salamon, P., Rohwer, F., 2003. Metagenomic analyses of an uncultured viral community from human feces. J. Bacteriol. 185 (20), 6220e6223. Cole, D., Long, S.C., Sobsey, M.D., 2003. Evaluation of Fþ RNA and DNA coliphages as source specific indicators of fecal contamination in surface waters. Appl. Environ. Microbiol. 69 (11), 6507e6514. Cornax, R., Morin˜igo, M.A., Gonzalez-Jaen, F., Alonso, M.C., Borrego, J.J., 1994. Bacteriophages presence in human feces of healthy subjects and patients with gastrointestinal disturbances. Zentralbl Bakteriol 281 (2), 214e224. Cox, C.R., Gilmore, M.S., 2007. Native microbial colonization of Drosophila and its use as a model of Enterococcus faecalis pathogenesis. Infect. Immun. 75 (4), 1565e1576. Devriese, L.A., Laurier, L., De Herdt, P., Haesebrouck, F., 1992. Enterococcal and streptococcal species isolated from feces ofcalves, young cattle and dairy cows. J. Appl. Bacteriol. 72 (1), 29e31. Duran, A.E., Muniesa, M., Mendez, X., Valero, F., Lucena, F., Jofre, J., 2001. Removal and inactivation of indicator bacteriophages in fresh waters. J. Appl. Microbiol. 92, 338e347. Field, K.G., Samadpour, M., 2007. Fecal source tracking, the indicator paradigm, and managing water quality. Water Res. 41 (16), 3517e3538. Fong, T.T., Lipp, E.K., 2005. Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools. Microbiol. Mol. Biol. Rev. 69 (2), 357e371. Classification and Nomenclature of Viruses. 1982. Fourth report of the International Committee on Taxonomy of Viruses in Intervirology 17(1e3), 1e199. Furuse, K., Ando, A., Osawa, S., Watanabe, I., 1981. Distribution of ribonucleic acid coliphages in raw sewage from treatment plants in Japan. Appl. Environ. Microbiol. 41 (5), 1139e1143. Furuse, K., Osawa, S., Kawashiro, J., Tanaka, R., Ozawa, A., Sawamura, S., Yanagawa, Y., Nagao, T., Watnabe, I., 1983. Bacteriophage distribution in human faeces: continuous survey of healthy subjects and patients with internal and leukaemic diseases. J. Gen. Virol. 64 (9), 2039e2043. Gantzer, C., Maul, A., Audic, J.M., Schwartzbrod, L., 1998. Detection of infectious enteroviruses, enterovirus genomes, somatic coliphages, and Bacteroides fragilis phages in treated wastewater. Appl. Environ. Microbiol. 64 (11), 4307e4312. Gantzer, C., Giller, L., Kuznetsov, M., Oron, G., 2001. Adsorption and survival of faecal coliforms, somatic coliphages and F-specific RNA phages in soil irrigated with wastewater. Water Sci. Technol. 43 (12), 117e124. Gantzer, C., Henny, J., Schwartzbrod, L., 2002. Bacteroides fragilis and Escherichia coli bacteriophages in human faeces. Int. J. Hyg. Environ. Health 205 (4), 325e328. Golomidova, A., Kulikov, E., Isaeva, A., Manykin, A., Letarov, A., 2007. The diversity of coliphages and coliforms in horse feces reveals a complex pattern of ecological interactions. Appl. Environ. Microbiol. 73 (19), 5975e5981. Grabow, W.O.K., Coubrough, P., 1986. Practical direct plaque assay for coliphages in 100 ml samples of drinking water. Appl. Environ. Microbiol. 52 (3), 430e433. Hernandez-Delgado, E.A., Sierra, M.L., Toranzos, G.A., 1991. Coliphages as alternate indicators of fecal contamination in tropical waters. Environ. Toxicol. Water Qual. 6, 131e143.

w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 7 1 6 e4 7 2 5

Hernandez-Delgado, E.A., Toranzos, G.A., 1995. In-situ replication studies of somatic and male-specific coliphages in a tropical pristine river. Water Sci. Technol. 31, 247e250. Huang, Z., Fasco, M.J., Kaminsky, L.S., 1996. Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. Biotechniques 20 (6), 1012e1014. 1016, 1018e1020. Hurst, C.J., 1996. Overview of water microbiology as it relates to public health. In: Crawford, R.L. (Ed.), Manual of Environmental Microbiology. ASM Press, pp. 219e222. Jofre, J., Olle, E., Ribas, F., Vidal, A., Lucena, F., 1995. Potential usefulness of bacteriophages that infect Bacteroides fragilis as model organisms for monitoring virus removal in drinking water treatment plants. Appl. Environ. Microbiol. 61 (9), 3227e3331. Kelly, S.M., Sanderson, W.W., 1960. The effect of chlorine in water on enteric viruses. II. The effect of combined chlorine on poliomyelitis and Coxsackie viruses. Am. J. Public Health Nations Health 50, 14e20. Keswick, B.H., Gerba, C.P., DuPont, H.L., Rose, J.B., 1984. Detection of enteric viruses in treated drinking water. Appl. Environ. Microbiol. 47 (6), 1290e1294. Keswick, B.H., Satterwhite, T.K., Johnson, P.C., DuPont, H.L., Secor, S.L., Bitsura, J.A., Gary, G.W., Hoff, J.C., 1985. Inactivation of Norwalk virus in drinking water by chlorine. Appl. Environ. Microbiol. 50 (2), 261e264. Khiyama, H.M., Makemson, J.C., 1973. Sand beach bacteria: enumeration and characterization. Appl. Microbiol. 26 (3), 293e297. Long, S.C., Sobsey, M.D., 2004. A comparison of the survival of FþRNA and FþDNA coliphages in lake water microcosms. J. Water Health 2 (1), 15e22. Moce-Llivina, L., Lucena, F., Jofre, J., 2005. Enteroviruses and bacteriophages in bathing waters. Appl. Environ. Microbiol. 71 (11), 6838e6844. Moe, C.L., 1996. Waterborne transmission of infectious Agents. In: Crawford, R.L. (Ed.), Manual of Environmental Microbiology. ASM Press, pp. 222e240. Moldovan, R., Chapman-McQuiston, E., Wu, X.L., 2007. On kinetics of phage adsorption. Biophys. J. 93, 303e315. Noble, R.T., Lee, I.M., Schiff, K.C., 2003. Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater. J. Appl. Microbiol. 96, 464e472. Noble, R.T., Fuhrman, J.A., 1996. Virus decay and its causes in coastal waters. Appl. Environ. Microbiol. 63 (1), 77e83. Payment, P., Franco, E., 1993. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water

4725

treatment for viruses and protozoan cysts. Appl. Environ. Microbiol. 59 (8), 2418e2424. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. RNase that is free of DNase. In: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Scott, T.M., Rose, J.B., Jenkins, T.M., Farrah, S.R., Lukasi, J., 2002. Microbial source tracking: current methodology and future directions. Appl. Environ. Microbiol. 68 (12), 5796e5803. Seeley, N.D., Primrose, S.B., 1980. The effect of temperature on the ecology of aquatic bacteriophages. J. Gen. Virol. 46, 87e95. Sinton, L.W., Hall, C., Braithwaite, R., 2007. Sunlight inactivation of Campylobacter jejuni and Salmonella enterica, compared with Escherichia coli, in seawater and river water. J. Water Health 5, 357e365. Sinton, L.W., Davies-Colley, R.J., Bell, R.G., 1994. Inactivation of enterococci and fecal coliforms from sewage and meatworks effluents in seawater chambers. Appl. Environ. Microbiol. 60 (6), 2040e2048. Stetler, R.E., 1984. Coliphages as indicators of enteroviruses. Appl. Environ. Microbiol. 48 (3), 668e670. Tavakoli, A., Yazdani, R., Shahmansouri, M.R., Isfahani, B.N., 2002. Chlorine residual efficiency in inactivating bacteria from secondary contamination in Isfahan. East Mediterr. Health J. 11 (3), 425e434. Toranzos, G.A., McFeters, G.A., Borrego, J.J., Savill, M., 1996. Detection of microorganisms in environmental freshwaters and drinking waters. In: Crawford, R.L. (Ed.), Manual of Environmental Microbiology. ASM Press, pp. 249e260. Uchiyama, J., Rachel, M., Maeda, Y., Takemura, I., Sugihara, S., Akechi, K., Muraoka, A., Wakiguchi, H., Matsuzaku, S., 2007. Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate. FEMS Microbiol. Lett. 278 (2), 200e206. USEPA, 2002. Method 1600: Membrane Filter Test Method for Enterococci in Water. EPA-821-R-02e022. Office of Water, Washington DC. Ward, R.L., Knowlton, D.R., Winston, P.E., 1986. Mechanism of inactivation of enteric viruses in fresh water. Appl. Environ. Microbiol. 52 (3), 450e459. Weisberg, S.B., Noble, R.T., Griffith, J.F., 1996. Microbial indicators of marine recreational water quality. In: Crawford, D.L. (Ed.), Manual of Environmental Microbiology. ASM Press, Washington, D.C, pp. 280e287. Wolf, H.W., 1972. The coliform count as a measure of water quality. In: Mitchell, T. (Ed.), Water Pollution Microbiology. Wiley-Interscience, New York, pp. 333e345.