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Coliphages as Alternate Indicators of Fecal Contamination in Tropical Waters E.A. HERNANDEZ-DELGADO, M.L. SIERRA, and G.A. TORANZOS Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico 00931 ABSTRACT Strong evidence has recently been found against the use of fecal coliforms as indicator organisms of fecal contamination in tropical waters due to their indigenous nature in pristine waters. There is a great need for the development of more rapid, accurate, and low-cost techniques for determining bacteriological water quality. Coliphages seem to be a n excellent alternative. We have developed a method to analyze large volumes ofpristine water, which we compared to a commonly used direct method. It involves the filtration of water through positively charged filters and a virus-elution step. The eluent is then mixed with culture medium, a host bacterium, and incubated. We sampled pristine tropical rivers, water colleeted from bromeliads (epiphytic vegetation), sewage-contaminated waters, and marine waters. Concentrations of indicator bacteria were higher than recommended levels for recreational waters, including bromeliad waters. Indicators levels were higher in bromeliad waters than in sewage-contaminated rivers. Phages were isolated only from waters being used for recreational purposes and from sites known to be contaminated with domestic sewage, but not from pristine or bromeliad waters. These results suggest that there is a correlation between the presence of coliphages and fecal contamination. This further suggests that coliphages may be reliable indicators of fecal contamination in the environment. INTRODUCTION Among some of the world’s greatest public health problems have been waterborne diseases, especially in tropical and subtropical areas, where the diversity and severity of these diseases is higher. Three groups of indicator microorganisms have been used to monitor water for the possible presence of enteric pathogens. However, evidence has been recently found against the use of fecal coliforms as indicator organisms of fecal contamination in tropical waters (Hazen, 1988). Several studies in rain forest watersheds of Puerto Rico indicated Environmental Toxicology and Water Quality: An International Journal Vol. 6, 131-143 (1991) CCC 1053-4725/91/020131-13$04.00 0 1991 John Wiley & Sons, Inc. 132/HERNANDEZ-DELGADOET AL. that Escherichia coli could survive, remain physiologically active, and grow in the environment (Carrillo et al., 1985;Hazen et al., 1987;L6pezTorres et al., 1987,1988; Santiago-Mercadoand Hazen, 1987;Bermudez and Hazen, 1988; Jim6nez et al., 1989; P6rez-Rosas and Hazen, 1989). These indicator organisms have also been isolated in other tropical countries from pristine waters (Feachem, 1974; Oluwande et al., 1983; Fujioka et al., 1988). See Hazen (1988) for a review. Fecal coliforms and fecal streptococci have also been isolated from water accumulated in the axilae of bromeliads (epiphytic flora) in a Puerto Rican tropical rain forest (Rivera et al., 1988). High natural productivity of tropical environments probably allows for survival and regrowth of pathogens and indicators in freshwater. In addition to the presence of indigenous indicators in the environment, these are probably only a few of the reasons why only 13 of the 67 river sampling stations (19%) failed to meet the maximum contaminant levels standards for recreational waters in recent years (less than 100 fecal coliforms/100 mL) (Curtis et al., 1984). None of the sites sampled met raw source water standards (less than 2 fecal coliforms/100 mL). There is a great need for the development of more rapid, accurate, and low-cost techniques for determining microbiological water quality. Coliphages have been proposed as an alternate and economically reliable indicator of fecal contamination of water (Wentsel et al., 1982; Grabow and Coubrough, 1986; Sim and Dutka, 1987; Castillo et al., 1988; Sim et al., 1988; Toranzos et al., 1988) because phage assays are easier and cheaper to perform than currently used bacteriological techniques. In addition, they can be quantified in environmental samples within 3-24 h as compared to at least 96 h when performing bacteriological assays. One of the many potential applications of bacteriophages is as indicators of sewage contamination (Gerba, 1987).The ubiquity of coliphages in the feces of warm-blood animals, sewage, and in sewagecontaminated waters suggests that coliphages could reliably indicate fecal contamination of the environment. Coliphages also exhibit a great resistance to environmental stress, and thus the longest survival of the bacteria and viruses excreted in the feces. They are, then, potentially useful for detecting remote sources of fecal contamination or to indicate fecal contamination of waters. Commonly used indicators, including E . coli, are naturally occurring in pristine waters, whereas coliphages have never been detected at these same sites, suggesting the absence of fecal contamination. In Puerto Rico coliphages have only been isolated from streams and rivers being used for recreational purposes, and from sewage-con- COLIPHAGES AS FECAL CONTAMINATION INDICATORS/ 133 taminated rivers, estuaries, and beaches, suggesting a correlation between the presence of phages and human fecal contamination. Although the use of coliphages as indicators of fecal contamination appears attractive, thorough studies involving statistical comparisons with the presence of other microbial indicators and pathogens, and environmental coliphage survival, are needed before they can be seriously considered as meaningful indicators of fecal contamination. The purpose of this study was to evaluate the reliability of coliphages as alternate indicators of fecal contamination in tropical surface freshand marine waters in Puerto Rico. MATERIALS AND METHODS Study Sites The Mameyes River watershed located in the Caribbean National Rain Forest was selected because it offers several different habitats. The river begins at an elevation of nearly 1000m above sea level. According to L6pez-Torres et al. (1987), the upper third is in the Caribbean National Rain Forest, and it is characterized by pristine waters in the upper parts and recreational waters in its lower area. The middle third receives primary treated domestic sewage from a large housing development and it is also used for recreation. The lower third passes through two towns, where it is further polluted by domestic and industrial sewage effluents. This portion of the river is commonly frequented by fishermen (personal observations). The river empties into the Atlantic Ocean at Fortuna Beach. It is located near Luquillo Beach, Puerto Rico’s largest public beach. Sampling Procedures Grab samples were collected for bacteriological and phage assays in 1L sterile Nalgene plastic containers (Nalge Company, Rochester, NY) and kept on ice until assayed. PHAGE assays were carried out within 6 h while bacteriological assays were conducted within 24 h. Bacteriological Analysis Samples were analyzed by the membrane filtration technique using m-Endoagar for the isolation of total coliforms, the m-FC medium for fecal coliforms, and KF-Streptococcus agar for fecal streptococci.Repre- 134/HERNANDEZ-DELGADOET AL. sentative colonies were confirmed using standard techniques (American Public Health Association, 1985). Bacteriophage Host An E . coli C strain (ATCC 15597) was utilized as the phage host. Previous studies with this strain have shown it to be the most sensitive host for the isolation of coliphages from the environment. Phage Assays A modified adsorption-elution technique was evaluated to isolate phages from pristine waters. This technique involves the filtration of large volumes (one liter or larger) of water through a positively charged filter (Versapor 30S, obtained from AMF-Cuno Division, Meriden, CT), where phages adsorb. Then, they were desorbed in a small volume of 3%beef extract + 3% tryptose phosphate broth (pH 8.0). This eluent was mixed with trypticase soy agar (Scott Laboratories, Fiskeville, RI) and the phage host cells, and incubated at 37°C for 12 h. Contaminated water samples were analyzed by a direct method (Grabow and Coubrough, 1986). Briefly explained: a 100-mL sample of water was mixed with 100 mL of trypticase soy agar (2 x concentration) and the host cell, then poured into sterile Petri dishes and incubated at 37°C for 12 h. Because of the low volume sampled, this is not an adequate assay for pristine waters, where a large volume is needed to detect low numbers of phages. Phage Survival Studies Under Laboratory Conditions Survival studies were carried out in the laboratory. Briefly explained: environmental waters were seeded with lo7 plaque-forming units (PFU) isolated from domestic sewage and the rate of inactivation followed by obtaining samples at increasing time intervals. Phages were detected as described above. In Situ Phage Survival Studies In situ phage survival studies were carried out in a pristine tropical rain forest river using virus environmental survival chambers (25 mL) loaded with 0.01- and 0.45-pm pore size filters. Phages isolated from raw sewage were mixed with sterile water (chamber A), and with river water containing natural coliform strains (chamber B). MS-2 were mixed with sterile water and used as a control (chamber C).Aliquots COLIPHAGES AS FECAL CONTAMINATION INDICATORSI135 TABLE I Average concentrationsa of coliphages and indicator bacteria in several sampling stations in the Mameyes River watershed, Puerto Rico Sampling station Bromeliad waterb La Mina River La Mina River (recreational area) La Coca Fall Mameyes River (Puente Roto) Mameyes River (posteffluent) Mameyes River estuary Fortuna Beach Total coliforms Fecal coliforms Fecal streptococci 1.42 x lo6 4.21 x lo4 8.37 x lo4 0 1.62 x 104 3.22 x 104 5.12 x 103 6.05 x 103 2.30 x 103 6.91 x 103 0 0.17 1.88 X lo3 3.63 x 104 4.66 X lo2 1.07 x 104 1.70 X lo3 6.81 x 103 1 2.14 2.12 x 104 9.09 x 103 2.14 x 104 8.5 1.94 x lo4 3.07 x lo4 1.35 x lo4 1.52 x lo2 3.34 x 104 2.11 x 104 2.00 x 101 Coliphages 37.8 a All concentrations of bacteria and phages are in colony-forming units (CFU)/100 mL and PFU/100 mL, respectively; averages calculated from 10 to 14 samples (about 2 samples/month). Sites arranged in order ranging from pristine to sewage-contaminated waters. were taken at different time intervals up to 20 days. River water temperature, pH, conductivity, hardness, dissolved oxygen, and turbidity were measured for each sampling time. RESULTS AND DISCUSSION We have analyzed pristine tropical rivers, water collected from bromeliads (epiphytic vegetation), sewage-contaminated waters, and marine waters. Total and fecal coliforms levels were almost always higher than recommended standards of less than 1000 fecal coliforms/100 mL for recreational waters in all samples. The same was true for fecal streptococci. None of the samples met raw source water standards of less than 2 fecal coliformsll00 mL. Results shown in Table I clearly indicate the natural presence of indicators in pristine waters as in the upper parts of the mountains in La Mina River and water from the axilae of bromeliads. In fact, bromeliad waters had higher average concentrations of total coliforms and fecal streptococci than surface waters. The highest levels of fecal coliforms were observed at the Mameyes River estuary, followed by water from the bromeliads. The lower levels of bacteria ND lo2 6.24 x 8.90 x lo3 9.0 0 0 6.5 ND ND ND Phages FS 1.69 x 103 2.21 x lo2 2.20 x 102 June 1989 FC Tntc 9.25 x lo2 4.10 x 103 TC 7.36 x 104 8.70 x 104 1.69 x 104 3.72 x 104 TC 4.66 x 103 1.08 x 103 1.16 x 103 7.53 x 102 FS 1.67 x 104 1.49 x 105 6.91 x 103 2.91 x 104 July 1989 FC 16.6 0 0 2.5 Phages a All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL, respectively; averages calculated from 2 samples/ month. Abbreviations: TC, total coliform; FC, fecal coliform; FS, fecal streptococci; ND, not done; Tntc, too numerous to count. Sites arranged in order ranging from pristine to sewage-contaminated waters. Bromeliad waterC La Mina River Mameyes River (Puente Roto) Mameyes River (posteffluent) Sampling station TABLE I1 Concentrationsa of coliphages and indicator bacteria in several sampling stations through the months of June and July 1989 in the Mameyes River watershed, Puerto Ricob *r m 0 5 z F U 35 8.70 x 104 ND ND 3.17 x 103 ND ND 1.01 x 105 ND ND Tntc Tntc 1.17 x 105 1.19 x 103 6.60 x 104 Tntc 2.08 x 104 1.00 x 104 8.00 x 102 2.20 x 103 ND Tntc 7.00 x 103 4.60 x 10' 1.03 x 103 3.00 x lo2 7.45 x 102 6.27 x lo2 1.46 x 5.39 x 8.5 0 0 0 0 ND lo2 102 Phages a All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL, respectively; averages calculated from 2 samples/ month. For abbreviations, see footnote b of Table 11, except for ND:not done because of a drought that followed Hurricane Hugo, water did not accumulate in the axilae of bromeliads. Sites arranged in order ranging from pristine to sewage-contaminated waters. ND ND 2 4 2.37 x 103 2.92 x 103 1.32 X 10' 2.92 x 103 2.56 x 103 2.69 x 104 ND 4.00 x 10' 1.50 x 103 ND 5.04 x 103 2.29 x 104 0 0 0.5 1.80 x 105 2.45 x 103 1.81 x 104 1.30 x 104 1.95 X 10' 3.54 x 102 1.11 x 106 1.40 x 104 3.18 x 104 Bromeliad water' La Mina River La Mina River (recreational area) La Coca Fall Mameyes River (Puente Roto) Mameyes River (posteffluent) Mameyes River estuary Fortuna Beach FS September 1989 FC TC Phages FS August 1989 station FC TC Sampling TABLE I11 Concentrations" of coliphages and indicator bacteria in several sampling stations through the months of August and September 1989 in the Mameyes River watershed, Puerto Ricob 4 w r 3 ij ND 3.00 x 105 ND ND ND ND ND x 103 4.00 x 104 4.00 1.90 x 103 ND 2.20 x 104 ND ND ND December 1989 FC FS 1.00 x 105 ND 1.00 x 105 TC 2 5 6 0 0 0 Phages ND ND 4.00 x 104 TC 5.35 x 106 ND 1.00 x 104 ND 6.00 x 105 3.05 x 104 1.90 x 105 ND 4.25 x 104 x 103 ND 2.00 x 104 2.00 LOO x 105 1.00 x 103 1.00 x 103 January 1990 FC FS 3 64 0 2 0 0 Phages a All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL respectively; averages calculated from 2 samples/ month. For abbreviations, see footnote b to Table 11. Sites arranged in order ranging from pristine to sewage-contaminated waters. Bromeliad watel" La Mina River Mameyes River (Puente Roto) Mameyes River (posteflluent) Mameyes River estuary Fortuna Beach Sampling station TABLE IV Concentrationsa of coliphages and indicator bacteria in several sampling stations through the months of December 1989 and January 1990 in the Mameyes River watershed, Puerto Ricob + *r B r3 0 E s* U E? 2 iN E ? w COLIPHAGES AS FECAL CONTAMINATION INDICATORS/ 139 TABLE V Concentrationsa of coliphages and indicator bacteria in several sampling stations in the month of March 1990 in the Mameyes River watershed, Puerto Ricob ~ Sampling station Bromeliad water' La Mina River La Mina River (recreational area) Mameyes River (Puente Roto) Mameyes River (posteffluent) Mameyes River estuary Fortuna Beach ~ ~ March 1990 TC FC FS Phages 1.42 x 105 4.43 x 104 4.20 x 104 1.60 x 104 2.36 x 104 1.63 x 104 4.10 2.00 x 103 X lo2 2.00 x 103 0 0 0 1.00 4.35 x 103 7.50 X lo2 0 3.15 x 104 1.07 x 104 6.00 x lo2 1 1.94 x 104 1.02 x 104 ND 1 3.34 x 104 2.11 x 104 2.00 x 10' 0 xi04 a All concentrations of bacteria and phages are in CFU/100 mL and PFUilOO mL, respectively; averages calculated from 2 samples/month. For abbreviations, see footnote b of Table 11. ' Sites arranged in order ranging from pristine to sewage-contaminated waters. were detected at La Coca Fall. Higher levels of indicator bacteria in the rivers coincided with heavy rainfall and turbidity (possibly due to stirring up of the sediments), while the opposite was observed in the bromeliads, which was probably due to dilution effects. Higher levels of bacteria from the bromeliads were detected during the dry season (i.e., March). These results are shown in Tables 11-V, which compare results between different months for a 10-month period. Coliphages were isolated only from recreational waters and from sites known to be contaminated with sewage, but not from bromeliad and pristine waters. Higher coliphage levels were observed at the Mameyes River estuary. These waters receive sewage effluents from two small towns and two primary treatment plants. This suggests that there may be a correlation between the presence of coliphages and fecal contamination, and further indicates that coliphages may be reliable indicators of fecal contamination in the environment. Coliphage survival studies under laboratory conditions indicated that total and fecal coliform levels increased over time in both pristine and sewage-contaminated waters (Tables VI and VII). Coliphage levels decreased 4 and 3 orders of magnitude for pristine and sewage-contaminated waters, respectively, after 120 h. In addition, the decrease in 140/HERNANDEZ-DELGADO ET AL. TABLE VI Coliphage survival under laboratory conditions in pristine river waters Coliforms b e r 100 mL)" Time (h) pH Total 0 24 48 72 120 7.0 6.3 5.6 4.2 3.3 4.7 x 104 2.6 x lo7 1.5 x los 1.7 x 10' 1.1 x 106 a Fecal 6.0 3.4 2.7 1.7 3.0 x 103 x lo6 x lo6 x lo6 x 104 Fecal streptococci (per 100 mL) ND~ 105 105 104 103 1.0 x 1.6 x 2.8 x 2.0 x Coliphages (per 100 mL) 7.0 2.3 6.0 9.0 3.0 x lo6 x 105 x 104 x 104 x lo2 Total and fecal coliforms and streptococci were naturally occurring in these waters. ND. not done. phage concentration was directly proportional to the decrease in the pH of both types of water. In order to evaluate phages' survival in surface freshwater, and to determine if they can replicate in natural coliform strains under environmental conditions, an in situ survival study was carried out in a pristine river. No coliphage inactivation was observed during the first 192 h (8 days) of the study (Fig. 1).This was probably due to their resistance to environmental factors. As environmental parameters did not fluctuate greatly through the study, they did not appear to have had a significant effect on phage stability. After 10 days, a 2 log decrease was observed in the sewage-isolated coliphage chamber mixed with river water containing natural coliform strains. It was demonstrated that sewage-bornecoliphages did not replicate under environmental conditions using indigenous E . coli as hosts. TABLE VII Coliphage survival under laboratory conditions in sewage-contaminated waters Coliforms (per 100 mL) Time (h) pH Total Fecal Fecal streptococci (per 100 mL) 0 24 7.0 6.6 6.0 5.4 5.4 1.7 X lo6 3.8 x lo7 1.1 x lo8 8.0 x 107 3.0 x lo6 1.1 x lo3 2.1 x lo6 2.7 x lo6 2.0 x 106 1.1 x lo6 ND" 2.0 x 105 9.0 x 104 4.4 x 104 2.1 x 104 48 72 120 a ND, not done. Coliphages (per 100 mL) 7.7 2.4 2.0 3.7 4.7 x x x x x 106 105 105 104 103 COLIPHAGES AS FECAL CONTAMINATION INDICATORS1141 101 109 - . E 108 a 10 U n CHAMBER A CHAMBER B CHAMBER C 106 10 10’ I . , 0 2 . , 4 ., ., . 6 8 , . , . , 10 12 14 1 , 36 . , 18 . , I 20 TIME (DAYS) Chamber A = Phages isolated from raw sewage + sterile water Chamber B = Phages isolated from raw sewage + river water containing indigenous fecal coliforms Chamber C = MS-2 Phage + sterile water Fig. 1. Survival of coliphages in a tropical rain forest pristine river. In previously published studies (Castillo et al., 1988; Grabow and Coubrough, 1986; Wentsel et al., 19821, all coliphage data were obtained from small sample volumes; thus if low concentrations of phages were present, they were not detected due to the low sensitivity of the technique. In our technique, the concentration of 1L of sample down to 50 mL, which can then be easily analyzed, improves on the ability to detect very low levels of phages. Nonetheless, even concentrating this volume, it was not possible to detect coliphages except in waters known to be contaminated with sewage or waters being used for recreational purposes. For our filtration-elution method, using water seeded with MS2, adsorption efficiencies ranging from 93 to 99.8%were obtained. A 43% elution efficiency was obtained using 3% beef extract + 3% tryptose phosphate broth (pH 8.0). The method has been tested in pristine river waters and in tap water samples, where up to 3 L of water were filtered. In the former, no coliphages were detected, while in the latter a total of 3 PFUiL were detected. We have detected coliphages only in sites where there is fecal contamination. Thus, coliphages seem to have the potential as an excellent alternative to the use of bacteria to indicate fecal contamination of waters and therefore to predict the possible presence of pathogens. l&/HERNANDEZ-DELGADO ET AL. CONCLUSIONS Total and fecal coliforms levels were always higher than recommended standards in all samples. They were also constantly isolated from pristine river waters and water from the axilae of bromeliads, where no human fecal contamination was detected. Coliphages were detected only in recreational waters and waters known to be sewage contaminated. They were never detected in pristine rivers. These results suggest an association of coliphages with human fecal contamination. Phage survival studies under laboratory conditions demonstrated that phage inactivation was directly proportional t o a decrease in pH, while in situ survival studies demonstrated that coliphages did not replicate in indigenous coliform strains under environmental conditions. Highest coliphage inactivation was observed in the sewageisolated coliphages mixed with river water containing resident flora. Coliphages have the potential of being used as indicators of fecal contamination in aquatic environments in spite of its long survival period of time, because its dilution in the environment will prevent their constant detection. In addition, phages did not replicate under laboratory or under environmental conditions in the presence of indigenous fecal coliform bacteria. Our filtration-elution method showed an adsorption efficiency ranging between 93 and 99.8%, and a 43% elution recovery. It proved to be an excellent method for phage concentration where phage levels were low (as in tap water) and where other methods (i.e., Grabow and Coubrough, 1 9 8 6 ) are less sensitive. Studies are being carried out on the detection of bacterial, viral, and parasitic pathogens in the environment. We are correlating them with the presence of coliphages. In addition, we are currently working on molecular techniques for the detection of coliphages in environmental samples, including the use of genetic probes and PCR techniques. We acknowledge the technical help of Nayah Harb and Leonor Alicea. Portions of this work were supported by SUBE no. 2S06RR08102-18, INDUNIV no. 9020539303, and WRRI 14-080001-G1611. References American Public Health Association. 1985. Standard Methods for the Examination of Water and Wastewater (16th edition). Washington, DC. Bermudez, M., and T.C. Hazen. 1988. Phenotypic and genotypic comparison ofEscherichia coEi from pristine tropical waters. Appl. Environ. Microbiol. 54:979-983. COLIPHAGES AS FECAL CONTAMINATION INDICATORS/ 143 Carrillo, M., E. Estrada, and T.C. Hazen. 1985. Survival and enumeration of the fecal indicators Bifidobacteriurn adolescentis and Escherichia coli in a tropical rain forest watershed. Appl. Environ. Microbiol. 50:468-476. Castillo, G., R. Thiers, B.J. Dutka, and A.H. 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