Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide

We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T₉₉ (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.     

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms

Natural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R₁ (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R₂ (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 °C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R₁ and R₂ were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known.     

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% host-specificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10⁻⁵, 10⁻⁴ and 10⁻³ respectively. HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10⁻⁴ whilst EVs were detected up to dilution 10⁻⁵. The ability of the HS-HF183 marker to detect fresh sewage was 3-4 orders of magnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.     

High diversity and differential persistence of fecal Bacteroidales population spiked into freshwater microcosm

Bacteroidales markers are promising indicators of fecal pollution and are now widely used in microbial source tracking (MST) studies. However, a thorough understanding of the persistence of Bacteroidales population after being released into environmental waters is lacking. We investigated the persistence of two host specific markers (HF183 and CF193) and temporal change of Bacteroidales population over 14 days in freshwater microcosms seeded with human or bovine feces. The concentrations of HF183/CF193 and Escherichia coli were determined using quantitative polymerase chain reaction (qPCR) and standard cultivation method, respectively. Shifts in the Bacteroidales population structure were fingerprinted using PCR-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing analysis targeting both 16S rDNA and rRNA-transcribed cDNA. Both HF183 and CF193 decayed significantly faster than E. coli but the decay curves fit poorly with first-order model. High diversity of Bacteroidales population was observed for both microcosms, and persistence of different species in the population varied. Sequence analysis indicated that most of the bovine Bacteroidales populations in our study are unexplored. DGGE and decay curve indicated that RNA decayed faster than DNA, further supporting the use of rRNA as indicator of metabolically active Bacteroidales population. Evaluations with more realistic scenarios are warranted prior to extending the results of this study to real field settings.     

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.     

Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces

We measured the concentrations of four host-specific (human, dog, cow, and horse Bacteroidales), four generic fecal (16S total Bacteroidales and Escherichia coli, 23S Enterococcus and uidA E. coli,) and two universal bacterial (16S universal and rpoB universal) DNA targets by qPCR in raw sewage and pooled fecal samples from dogs, cows, horses, and Canada Geese. A spiking protocol using the non-fecal bacterium Pseudomonas syringae pph6 was developed to estimate the recovery of DNA from fecal and environmental samples. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host type. The host-specific markers were found in high concentrations (8-9 log₁₀ copies/g dry wt.) in their respective hosts' samples, which were equal to or greater than the concentrations of generic E. coli and Enterococcus markers, lending support to the use of host-specific and generic Bacteroidales as sensitive indicators of fecal pollution. The host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4% and horse-specific 2%. Based on this limited data set, the measurement of host-specific indicators by qPCR has several promising applications. These applications include determining the percentage of total Bacteroidales contributed by a specific host type, using the ratios of host-specific markers to E. coli or Enterococcus to estimate the contribution of each source to these regulated fecal indicator bacteria, and estimating the mass of feces from each host type in environmental samples.     

Lack of correlation between enterococcal counts and the presence of human specific fecal markers in Mississippi creek and coastal waters

The objective of this study was to determine whether statistically valid correlations could be shown between enterococcal counts of samples from creek and coastal sites and the presence of two molecular, library-independent markers that specify human and/or sewage pollution. Four hundred ninety samples were collected between August 2007 and April 2009 to determine enterococcal counts and the presence of genetic markers for the sewage indicator organisms Methanobrevibacter smithii and Bacteroidales. The presence of human/sewage markers and enterococcal counts were higher in creek samples than coastal samples, but the higher creek levels did not statistically correlate with the either enterococcal count or the presence of the markers present in coastal samples. Furthermore, there was no correlation between enterococcal counts in coastal samples and either marker at any of the beach sites tested. The results of this investigation in Mississippi coastal waters suggest that human/sewage markers are unlikely to correlate with enterococci counts in the nearshore environment and that enterococcal counts may be indicative of other animal or environmental sources. Additionally, a study comparing conventional gel electrophoresis with capillary electrophoresis did not convincingly establish that one method was better than the other in regard to the results obtained. The capillary method does allow reproducibility of results and the ability to analyze multiple samples in a short period of time; however, the operational expenditures exceed the cost of traditional gel electrophoresis.     

The enterovirus test in the assessment of recreational water-associated gastroenteritis

This study reports the results of a series of analyses designed to identify possible mathematical relationships between the numbers of fecal streptococci vs the numbers of enteroviruses present (as measured by the enterovirus assay) in marine recreational waters in order to indirectly assess the relationship between enteroviral densities and subsequent risk of gastroenteritis among bathers exposed to marine waters contaminated with domestic sewage. A database consisting of 2066 parallel fecal streptococci and enterovirus enumerations taken from 416 different marine water locations throughout the United Kingdom was used in the analyses. Polychotomous logistic regression was used to model the probabilities of zero enterovirus, a possible infectious dose and a probable infectious dose of enterovirus being present over increasing fecal streptococci densities. The results of the polychotomous logistic regression analyses showed that the probability of the absence of enterovirus (i.e., zero enteroviruses being present) remained higher than the probability of the presence of either a possible or probable infectious dose in 101 of seawater up to a fecal streptococci density of 1000/100 ml of sample (actual estimated probabilities for the three groupings of enterovirus densities described above, at a fecal streptococci density of 1000 equaled 0.44, 0.44, and 0.12, respectively). In addition, the probability of zero enteroviruses being present remained higher than the probability of one or more enteroviruses being present in 101 of seawater up to a fecal streptococci density of 450/100 ml of sample. Since previously published epidemiological studies have shown an excess risk of gastroenteritis to occur among bathers exposed to as few as 32 fecal streptococci per 100 ml of sample, these results suggest that the actual viruses enumerated by the enterovirus assay are not etiologically related to recreational water-associated gastroenteritis. Since gastroenteritis has been the most consistently reported bathing water associated illness, these findings suggest the enterovirus assay to be of limited use in assessing marine recreational water quality given the increasing tendency to formulate water quality standards based on increased risk of gastroenteritis among bathers relative to non-bathers.     

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.     

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.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers – Is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.     

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.     

Recovery of viruses from waste-water and effluent by the direct inoculation method

Chloroform or antibiotic treated sewage and effluent samples were inoculated directly onto MK₂ or Vero cell monolayers for enterovirus recovery. The results obtained showed a high enterovirus recovery. The average PFU (100 ml)⁻¹ from sewage samples was 4496 and from effluent samples was 5684. The conventional ratio of enteroviruses to coliforms in sewage is considered to be 1:65,000. By the direct inoculation method the ratio obtained was 1: 20,000 for sewage and 1: 2000 for effluent. It is believed the number of enteroviruses in sewage to be even higher than the ones obtained.     

Host-specific 16S rRNA gene markers of Bacteroidales for source tracking of fecal pollution in the subtropical coastal seawater of Hong Kong

This study investigated the diversity of Bacteroidales communities in the feces of eight host species in Hong Kong (subtropical Asia), including human (in the form of sewage), cow, pig, horse, cat, dog, rabbit and rat. The analysis of terminal restriction fragment length polymorphism (TRFLP) in the 16S rRNA genes revealed significant differences in Bacteroidales communities among all host species, with the exception of dog and cat. Manual examination of TRFLP profiles resulted in six terminal restriction fragments (TRFs) that were potentially specific to the sewage (one TRF), cow (three TRFs) or pig (two TRFs) samples. All six TRFs were (1) present in 100% of the samples of the respective target host, (2) absent in other hosts or present only in low frequency and low intensity, and (3) verified for sizes using in silico digestion of DNA sequences in clone libraries. The six TRFs could reliably indicate the source of fecal contaminations in natural seawater amended with sewage, cow or pig fecal samples. In field tests conducted for two polluted and one unpolluted coastal site, the sewage-specific TRF was detected in all seawater samples of the sites known to be impacted by raw and treated sewage. However, only two of three cow-specific TRFs were detected for the two polluted sites, which also received fecal input from feral cows. No pig-specific TRF was detected, although one of the coastal sites was chronically polluted by pig farm run-offs. Nevertheless, the total absence of the six potentially host-specific TRFs in the seawater of an unpolluted site demonstrated the specificity of the TRFs as gene markers in indicating actual pollution.     

Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.     

Persistence of host-associated Bacteroidales gene markers and their quantitative detection in an urban and agricultural mixed prairie watershed

Microbial source tracking is an emerging tool developed to protect water sources from faecal pollution. In this study, we evaluated the suitability of real time-quantitative PCR (qPCR) Taqman assays developed for detection of host-associated Bacteroidales markers in a prairie watershed. The qPCR primers and probes used in this study exhibited high accuracy (88-96% sensitivity and ≥99% host specificity) in detecting Bacteroidales spp. that are associated with faeces from humans, ruminants, bovines, and horses. The ruminant- and human-associated markers were also found in high concentrations within individual faecal samples, ranging from 3.4 to 7.3 log₁₀ marker copy number g⁻¹ of individual host faeces. Following validation of host sensitivity and specificity, the host-associated Bacteroidales markers were detected in the Qu’Appelle Valley watershed of Saskatchewan, Canada which experiences a diversity of anthropogenic inputs. Concentrations of the ruminant marker were well-correlated with proximity to cattle operations and there was a correlation between the marker and Escherichia coli concentrations at these sites. Low concentrations of the human faecal marker were measured throughout the sampling sites, and may indicate a consistent influx of human faecal pollution into the watershed area. Persistence of each of the Bacteroidales host-associated marker was also studied in situ. The results indicated that the markers persist for shorter periods of time (99% decay in <8 days) compared with the conventional E. coli marker (99% decay in >15 days), suggesting they are effective at detecting recent faecal contamination events. The levels of Bacteroidales markers and E. coli counts did not correlate with the presence of the pathogenic bacteria, Salmonella spp. or Campylobacter spp. detected in the Qu’Appelle Valley. Collectively, the results obtained in this study demonstrated that the qPCR approach for detecting host-associated Bacteroidales spp. markers can be a useful tool in helping to determine host-specific impacts of faecal pollution into a prairie watershed.     

Sourcing faecal pollution: a combination of library-dependent and library-independent methods to identify human faecal pollution in non-sewered catchments

Library-dependent (LD) (biochemical fingerprinting of Escherichia coli and enterococci) and library-independent (LI) (PCR detection of human-specific biomarkers) methods were used to detect human faecal pollution in three non-sewered catchments. In all, 550 E. coli isolates and 700 enterococci isolates were biochemically fingerprinted from 18 water samples and compared with metabolic fingerprint libraries of 4508 E. coli and 4833 enterococci isolates. E. coli fingerprints identified human unique biochemical phenotypes (BPTs) in nine out of 18 water samples; similarly, enterococci fingerprints identified human faecal pollution in 10 water samples. Seven samples were tested by PCR for the detection of biomarkers. Human-specific HF134 Bacteroides and enterococci surface protein (esp) biomarkers were detected in five samples. Four samples were also positive for HF183 Bacteroides biomarker. The combination of biomarkers detected human faecal pollution in six out of seven water samples. Of the seven samples analysed for both the indicators/markers, at least one indicator/marker was detected in every sample. Four of the seven PCR-positive samples were also positive for one of the human-specific E. coli or enterococci BPTs. The results indicated human faecal pollution in the studied sub-catchments after storm events. LD and LI methods used in this study complimented each other and provided additional information regarding the polluting sources when one method failed to detect human faecal pollution. Therefore, it is recommended that a combination of methods should be used to identify the source(s) of faecal pollution where possible.     

Airborne enteric coliphages and bacteria in sewage treatment plants

The concentrations of airborne culturable microorganisms were determined in wastewater and sludge treatment processes of seven sewage treatment plants. Two types of coliphages, Salmonella and total viable bacteria were sampled by the BioSampler and the numbers of faecal coliforms and enterococci were obtained from the Andersen 6-stage impactor. The BioSampler recovered higher numbers of airborne coliphage viruses than has been measured with other liquid samplers in previous studies, suggesting that this sampler has improved efficiency for sampling airborne coliphages. Airborne coliphages were detected in many stages of the wastewater or sludge treatment process. The highest microbiological air contaminations were found in pre-treatment and aerated grit separation stages of the operation. This was attributed to aerosolisation of microorganisms by mechanical handling or forced aeration. Aeration and settling processes located outdoors caused low microbial concentrations, but the brush aerator released more microorganisms into the air. Our results emphasize the necessity for controlling the exposure of sewage workers to airborne microorganisms, especially in process areas that involve mechanical agitation or forced aeration of wastewater.     

Decay of intestinal enterococci concentrations in high-energy estuarine and coastal waters: towards real-time T90 values for modelling faecal indicators in recreational waters

Intestinal enterococci are the principal 'health-evidence-based' parameter recommended by WHO for the assessment of marine recreational water compliance. Understanding the survival characteristics of these organisms in nearshore waters is central to public health protection using robust modelling to effect real-time prediction of water quality at recreation sites as recently suggested by WHO and the Commission of the European Communities Previous models have more often focused on the coliform parameters and assumed two static day-time and night-time T90 values to characterise the decay process. The principal driver for enterococci survival is the received dose of irradiance from sunlight. In the water column, transmission of irradiance is determined by turbidity produced by suspended material. This paper reports the results of irradiated microcosm experiments using simulated sunlight to investigate the decay of intestinal enterococci in relatively turbid estuarine and coastal waters collected from the Severn Estuary and Bristol Channel, UK. High-turbidity estuarine waters produced a T90 value of 39.5 h. Low-turbidity coastal waters produced a much shorter T90 value of 6.6 h. In experiments receiving no irradiation, high-turbidity estuarine waters also produced a longer T90 of 65.1 h compared with corresponding low-turbidity coastal waters, T90 24.8 h. Irradiated T90 values were correlated with salinity, turbidity and suspended solids (r>0.8, p<0.001). The results suggest that enterococci decay in irradiated experiments with turbidity >200 NTU is similar to decay observed under dark conditions. Most significantly, these results suggest that modelling turbidity and or suspended solids offers a potential means of predicting T90 values in 'real-time' for discrete cells of a hydrodynamic model.