Differential persistence of F-specific RNA phage subgroups hinders their use as single tracers for faecal source tracking in surface water

The four subgroups of F-specific RNA bacteriophages (I-IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated. The proportions of the F-specific RNA bacteriophage subgroups were measured in wastewaters, after inactivation in surface waters or after wastewater treatment and in mixtures of wastewater of human and animal origin. Our results indicate that phage groups differ in their persistence in the environment and to different disinfecting treatments. The greater survival of subgroups I and II in treated samples hinders the interpretation of results obtained with F-specific RNA bacteriophages. The phages of subgroups III and IV were the least resistant to all treatments. These results should be considered when using genotypes of F-specific RNA as sole tracers for faecal source tracking.     

Application of real-time PCR assays to genotyping of F-specific phages in river water and sediments in Japan

Genotyping of F-specific RNA phages is currently one of the most promising approaches to differentiate between human and animal fecal contamination in aquatic environments. In this study, a total of 18 river water and sediment samples were collected from the Tonegawa River basin, Japan, in order to describe the genogroup distribution of F-specific RNA and DNA phages using genogroup-specific real-time PCR assays. F-specific phages were detected in nine (100%) river water and six (67%) sediment samples. Eighty-five phage plaques were isolated from these samples and subjected to real-time PCR assays specific for the phages. F-specific RNA phages of human genogroups (II and III) were detected in 32 (38%) plaques, whereas those of animal genogroups (I and IV) were detected in 17 (20%) plaques. No correlation was observed between the genogroup distribution of F-specific RNA phages and the occurrence of human adenovirus genomes, suggesting that genotyping of the phages alone is inadequate for the evaluation of the occurrence of viruses in aquatic environments. SYBR Green-based real-time PCR assay revealed the presence of F-specific DNA phages in four (5%) plaques, which were further classified into two genogroups (fd- and f1-like phages) by sequence analysis. Thirty-two (38%) plaques were not classified as the F-specific phage genogroups, indicating the limited applicability of these real-time PCR assays to a wide range of aquatic environmental samples worldwide.     

The limitation of the ratio of fecal coliforms to total coliphage as a water pollution index

The fecal coliform populations of raw sewage, sewage lagoon effluent, and river water were determined using the most probable number technique. The total coliphage populations of the three water sources were determined using Escherichia coli B (ATCC 11303-1) host cells. The ratios of fecal coliforms to coliphage in the three water samples were 87:1, 4.2:1, and 0.15:1, respectively. The ratio of fecal coliforms to coliphage in stored raw sewage decreased from 87:1 to about 1:1 within 7 days at 20°C and within 28 days at 4°C. These changing ratios resulted from the greater longevity of the coliphage compared with that of the coliform bacteria. The use of the ratio of fecal coliforms to coliphage is not considered reliable as an index of when a fecal pollution event occurred because the ratio is influenced by prior contamination, presence of sediment, chlorination, and temperature.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis

Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.     

Characterization of sewage solid-associated viruses and behavior in natural waters

The association of bacteriophages and animal viruses with solids has been demonstrated to have a protective effect, resulting in enhanced survival in natural waters and resistance to inactivation by chlorine. In this study, attempts were made to differentiate solid-associated viruses and freely suspended viruses in secondarily treated sewage by the retention of sewage solids on membrane filters treated with fetal calf serum to prevent adsorption of freely suspended virus. Solid-associated viruses collected on membrane filters were eluted with pH 11.5, 0.05 M glycine buffer. The percentage of the total coliphage and animal virus associated with solids in secondarily treated sewage discharges ranged from < 1.0 to 24% and 3 to 100%, respectively. The largest quantity of solid-associated coliphage was attached to particles greater than 8.0 μm and less than 0.65 μm in size. Tapwater, lake water and estuarine water were all capable of eluting solid-associated coliphages. Elution of coliphages in marine water appeared to be related to the salinity of the water. Coliphages eluted from sewage solids in seawater could readsorb to naturally occurring marine sediment.     

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

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic 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 by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and 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.     

Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe

Somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. We tested fresh and marine bathing waters from North, South, East and West Europe expected to contain between 100 and 500 E. coli per 100 ml, although wider ranges were sometimes found. Bacteriophages were counted after concentration, since a preliminary study proved that this step was necessary to obtain positive counts. During monitoring, a first-line quality control with reference materials for bacteria and bacteriophages was performed by all the laboratories participating in the study. The same microbes were also counted in raw sewage samples from various areas in Europe, where the bacterial indicators and the three groups of bacteriophages were detected in roughly the same numbers. All groups of bacteriophages were detected in both fresh and marine bathing waters throughout Europe. Reliable and complete results from 147 samples showed that for log-transformed values, E. coli and bacteriophages were slightly correlated. However, the slope of the regression line changed according to E. coli concentration and the correlation diminished when this concentration was close to zero per 100 ml. The ratios between E. coli and phages in bathing waters differed significantly from those in sewage. The relative amounts of bacteriophages, mainly somatic coliphages and phages infecting Bact. fragilis RYC2056, increased in bathing waters with low E. coli concentration, especially in seawater samples containing <100 E. coli per 100 ml. The relationship of bacteriophages with respect to enterococci paralleled that of bacteriophages with respect to E. coli. Somatic coliphages and bacteriophages infecting Bact. fragilis are useful to predict the presence of some pathogens with the same origin as present bacterial indicators but with higher survival rates.     

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory,comparative study

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10–50 gene copies or plaques × 50 ml⁻¹) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40–70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.     

Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays

Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and animal health.     

North and south american studies on the potential of coliphage as a water quality indicator

Studies were undertaken to assess the potential of coliphages to be used universally as water quality indicators and more specifically as health hazard indicators. Data were obtained from three water bodies, a northern Canadian River, inshore water samples from Lake Ontario and from marine beaches in Brazil. Data from this two continent, three water body study indicated (a) that within location fecal coliform and coliphages are positively correlated, (b) coliphage values can be predicted by using fecal coliform MPN, fecal streptococci MF and E. coli MF data and (c) a water quality guideline of 20 coliphage/100 ml for recreational fresh waters is proposed.     

Coliphages as indicators of enteric viruses in shellfish and shellfish raising estuarine waters

The practicality of a coliphage indicator system for human enterovirus in polluted waters was studied. Parallel examinations of sewage effluents, shellfish and shellfish growing waters for coliphage and enteric virus indicated a wide dissemination of coliphage throughout the estuary, generally occurring in the absence of detectable enteric virus activity. A majority of the enteric virus isolations were observed in samples yielding no coliphage activity. Under controlled conditions, oysters were observed to accumulate more coliphage than enteric virus. Replication of coliphage in the estuary during the summer months was shown to occur when proper host cell was present. Two major coliphage types were observed in field samples based on their reactivity with different Escherichia coli strains. A shift in dominant coliphage type was seen to occur during the study. Survival times of coliphage and enteric virus in estuary waters along with retention values in oysters were shown to be similar with a slight advantage shown by coliphage.Inability to correlate accurately coliphage and enteric virus occurrence in field samples along with the potential for the presence of more than one dominant coliphage type indicated the serious shortcomings of the coliphage indicator system as a method of enteric virus detection, leaving its overall use in the polluted environment subject to grave doubt.     

Antimicrobial resistance of fecal bacteria in waters of the Seine river watershed (France)

We studied the prevalences of antimicrobial resistance (AR) and multiple antimicrobial resistance (MAR) among the fecal bacteria found in the rivers of a large watershed under strong anthropogenic pressures, the Seine river watershed (France). Two groups of fecal indicator bacteria, Escherichia coli and intestinal enterococci, were tested for their susceptibility to 16 and 10 antimicrobials respectively, using the disk diffusion method. We found that 42% of the 214 E. coli river isolates were AR (resistant to at least one antimicrobial) and 35% were MAR (resistant to at least two antimicrobials). Among the 148 intestinal enterococci isolates from rivers, 83% were AR and 49% were MAR. We also investigated the sources of AR fecal bacteria found in the rivers of the watershed. A total of 715 E. coli isolates and 476 intestinal enterococci isolates were collected in point sources (municipal and hospital wastewaters) and non-point sources (surface runoff and soil leaching waters from agricultural or forest areas). For E. coli, the prevalence of AR differed widely from source to source and ranked in this order: hospital wastewaters (71%) > municipal wastewaters (44%) > agricultural non-point sources (16%) > forest non-point sources (2%). The prevalence of MAR ranked similarly, and the same trend was observed for intestinal enterococci. The AR level of fecal bacteria in the sources was related to their expected exposure level to antimicrobials before their release into the environment. A MAR index was calculated for every source and a good discrimination between them was thus obtained. At the global scale of the Seine river watershed, domestic wastewaters seemed more likely to be the predominant source of the AR fecal bacteria found in the rivers. This was corroborated by the similarity of the MAR indices from river and municipal wastewater isolates for both fecal indicators.     

Evaluation of multiple sewage-associated Bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for the detection of sewage pollution in environmental waters in Australia. The HuBac (i.e., 63%) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.     

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria were investigated in two large-scale municipal treatment plants during a period of six months. Total and relative numbers of resistant bacteria were determined in raw sewage, treated sewage and anaerobically digested sludge by bacteriological counts on media selective for coliforms (MacConkey agar) and Acinetobacter spp. (Baumann agar). In addition, the level of antimicrobial susceptibility was determined by the disc-diffusion method in 442 Acinetobacter isolates identified by colony hybridisation with a genus-specific DNA probe. Independent of the different antibiotics and media used, the total numbers of resistant bacteria in treated sewage were 10-1000 times lower than in raw sewage. Based on linear regression analysis of data on bacteriological counts, the prevalences of antimicrobial-resistant presumptive coliforms and Acinetobacter spp. in treated sewage and digested sludge were not significantly higher compared with raw sewage. On the contrary at one plant, statistically significant decreases were observed in the prevalence of ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0188) following sewage treatment, and in the prevalence of either ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0013) or ampicillin- and gentamicin-resistant presumptive coliforms (p = 0.0273 and p = 0.0186) following sludge treatment. The results obtained by bacteriological counts were confirmed by antimicrobial susceptibility testing of Acinetobacter isolates. Based on logistic regression analysis, isolates from treated sewage and digested sludge were generally not significantly more resistant compared with isolates from raw sewage. Based on these evidences, it was concluded that tertiary wastewater treatment did not result in a selection of antimicrobial resistant bacteria.     

Viruses and bacteriophages

Many of the enteric viruses which are transmitted from person to person by thefecal-oral route are found in raw and treated wastewater, and because of their persistence under adverse conditions may also be found in slightly polluted waters.There is no routine examination procedure of water and wastewater for entero-viruses,mainly because of the cumbersome isolation techniques, high cost and the need for highly skilled laboratory personnel.Phages are specific to single species of bacteria, are known for many entericbacteria, and are very often used for final identification of enteric pathogenic bacteria.Coliphages are provalent in raw and treated sewage as well as in polluted water, where enteric viruses may also be found.Coliphages were often mentioned as possible viral indicators in polluted water.To be a perfect indicator, they should comply with minimum criteria as follows: (a) they should be present wherever human enteric viruses are present: (b) the coliphage numbers recovered should be equal to or larger than those of enteric viruses recovered: (c) the coliphages should be at least as resistant as enteric viruses to adverse environmental conditions: (d) isolation and quantification of the coliphage should be faster and less expensive than isolation of the enteroviruses.Comparative studies show that the coliphage to enterovirus ratio in wastewater is about 10³:1. Levels of poliovirus 1 (attenuated) to coliphage f2 remained stable for a few months in oxidation pond effluents.f2 coliphage exhibited higher resistance to chlorination than poliovirus 1 (attenuated). When the two strains were kept in water of different quality, f2 survived longer. In addition, all coliphage counts were completed within 24 h. while those of enteroviruses required about a week. Results indicate very strongly that coliphages can be used as viral indicators and this is already the practice in a few European and other countries.     

Use of eukaryotic mitochondrial DNA to differentiate human, bovine, porcine and ovine sources in fecally contaminated surface water

A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.     

Alternative estimate of source distribution in microbial source tracking using posterior probabilities

Microbial source tracking (MST) is a procedure used to determine the relative contributions of humans and animals to fecal microbial contamination of surface waters in a given watershed. Studies of MST methodology have focused on optimizing sampling, laboratory, and statistical analysis methods in order to improve the reliability of determining which sources contributed most to surface water fecal contaminant. The usual approach for estimating a source distribution of microbial contamination is to classify water sample microbial isolates into discrete source categories and calculate the proportion of these isolates in each source category. The set of proportions is an estimate of the contaminant source distribution. In this paper we propose and compare an alternative method for estimating a source distribution—averaging posterior probabilities of source identity across isolates. We conducted a Monte Carlo simulation covering a wide variety of watershed scenarios to compare the two methods. The results show that averaging source posterior probabilities across isolates leads to more accurate source distribution estimates than proportions that follow classification.