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.     

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.     

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.     

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.     

Impact of urbanization and agriculture on the occurrence of bacterial pathogens and stx genes in coastal waterbodies of central California

Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and salinity.     

Evaluating the operational utility of a Bacteroidales quantitative PCR-based MST approach in determining the source of faecal indicator organisms at a UK bathing water

Microbial source tracking techniques are used in the UK to provide an evidence-base to guide major expenditure decisions and/or regulatory action relating to sewage disposal. Consequently, it is imperative that the techniques used robustly index faecal indicator organisms (FIOs) that are the regulatory parameters for bathing and shellfish harvesting areas. This study reports a ‘field-scale’ test of microbial source tracking (MST) based on the quantitative PCR analyses of Bacteroidales 16S rRNA genetic marker sequences. The project acquired data to test the operational utility of quantitative Bacteroidales MST data, comparing it with FIO concentrations in streams, effluents and bathing waters. Overall, the data did not exhibit a consistent pattern of significant correlations between Bacteroidales MST parameters and FIOs within the different sample matrices (i.e. rivers, bathing waters and/or effluents). Consequently, there was little evidence from this study that reported concentrations and/or percentages of human and/or ruminant faecal loadings (that are based on Bacteroidales MST gene copy numbers) offer a credible evidence-base describing FIO contributions to receiving water ‘non-compliance’. The study also showed (i) there was no significant attenuation of the Bacteroidales gene copy number ‘signal’ through the UV disinfection process; and (ii) single non-compliant samples submitted for Bacteroidales MST analysis, do not reliably characterise the balance of faecal loadings due to the high variability in the MST signal observed.At this stage in the development of the MST tool deployed, it would be imprudent to use the percentage human and/or ruminant contributions (i.e. as indicated by MST data acquired at a bathing water) as the sole or principal element in the evidence-base used to guide major expenditure decisions and/or regulatory action.     

Recommendations following a multi-laboratory comparison of microbial source tracking methods

Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero C_t values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions.     

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5β-cholestan-3β-ol (coprostanol), 5β-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5β-cholestan-3β-ol (24-ethylcoprostanol) and 24-ethyl-5β-cholestan-3α-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in “Microbial Source Tracking (MST)” methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of “pure” human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.     

Evaluating short-term changes in recreational water quality during a hydrograph event using a combination of microbial tracers, environmental microbiology, microbial source tracking and hydrological techniques: A case study in Southwest Wales, UK

Quantitative assessment of multiple sources to short-term variations in recreational water quality, as indexed by faecal indicator organism (FIO) concentrations, is becoming increasingly important with adoption of modern water quality standards and catchment-based water quality management requirements (e.g. the EU Water Framework Directive, Article 11 ‘Programmes of Measures’ and the US Clean Water Act, ‘Total Maximum Daily Loads’). This paper describes a study combining microbial tracers, intensive FIO measurement, open channel hydrology and molecular microbial source tracking (MST) to enhance understanding of recreational water quality at Amroth in southwest Wales, UK. Microbial tracers were released from four stream inputs during a moderate hydrograph event. Tracers from two local streams impacted simultaneously with a period of maximum FIO concentrations at the near-shore compliance monitoring site. Connection between these inputs and this site were rapid (9-33 min). Water quality impairment from a more remote stream input followed, 12.85 h after tracer release, sustaining FIO concentrations above desired compliance levels. MST analysis showed dominance of ruminant Bacteroidales genetic markers, associated with agricultural pollution. This integration of tracers and MST offers additional information on the movement and individual sources causing water quality impairment.     

The application of a recently isolated strain of Bacteroides (GB-124) to identify human sources of faecal pollution in a temperate river catchment

Recent work has suggested that bacteriophages infecting Bacteroides are a potential tool for faecal source tracking, but that different host strains may be needed for different geographic areas. This study used a recently identified strain of Bacteroides (GB-124) to detect human sources of faecal pollution in a river catchment in southeast England (UK). A total of 306 river water, municipal wastewater and animal samples were obtained over a 16-month period. Bacteriophages capable of infecting GB-124 were present in all municipal wastewaters but were not detected in faecal samples from animals, and were detected at significantly lower levels (P< 0.001) in river waters directly downstream of a dairy farm. This last observation was despite the presence of high levels of faecal indicator bacteria at this site. The study suggests that GB-124 appears to be specific to human faeces. As such it may represent an effective and low-cost method of faecal source identification.     

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.     

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.     

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.     

Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River watershed, Alberta, Canada

Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from 898 faecal, 43 sewage, and 342 surface water samples from the Oldman River were characterized using bacterial subtyping methods in order to investigate potential sources of contamination of the watershed. Among these pathogens, Campylobacter spp. were the most frequently isolated from faecal, sewage, and surface water samples (266/895, 11/43, and 91/342, respectively), followed by Salmonella (67/898, 8/43, and 29/342, respectively), and E. coli O157:H7 (16/898, 2/43, and 8/342, respectively). Salmonella Rubislaw was the most common serovar isolated from water. This serovar was also isolated from two wild bird species. Most other serovars isolated from water were either not isolated from animals or were isolated from multiple species. E. coli O157:H7 was predominantly isolated from cattle. The most common phage-types of this pathogen from cattle were also the most common among water isolates, and there were exact pulsed field gel electrophoresis and comparative genomic fingerprint matches between cattle, sewage, and water isolates. Campylobacters were commonly isolated from surface waters and faeces from most animal species. Restriction fragment length polymorphism of the Campylobacter flaA gene identified several location and host species-specific (cattle, goose, pig) fingerprints. Molecular subtyping of these bacterial pathogens shows considerable promise as a tool for determining the sources of faecal pollution of water.     

Integrated analysis of water quality parameters for cost-effective faecal pollution management in river catchments

In many parts of the world, microbial contamination of surface waters used for drinking, recreation, and shellfishery remains a pervasive risk to human health, especially in Less Economically Developed Countries (LEDC). However, the capacity to provide effective management strategies to break the waterborne route to human infection is often thwarted by our inability to identify the source of microbial contamination. Microbial Source Tracking (MST) has potential to improve water quality management in complex river catchments that are either routinely, or intermittently contaminated by faecal material from one or more sources, by attributing faecal loads to their human or non-human sources, and thereby supporting more rational approaches to microbial risk assessment. The River Ouse catchment in southeast England (U.K.) was used as a model with which to investigate the integration and application of a novel and simple MST approach to monitor microbial water quality over one calendar year, thereby encompassing a range of meteorological conditions. A key objective of the work was to develop simple low-cost protocols that could be easily replicated. Bacteriophages (viruses) capable of infecting a human specific strain of Bacteroides GB-124, and their correlation with presumptive Escherichia coli, were used to distinguish sources of faecal pollution. The results reported here suggest that in this river catchment the principal source of faecal pollution in most instances was non-human in origin. During storm events, presumptive E. coli and presumptive intestinal enterococci levels were 1.1-1.2 logs higher than during dry weather conditions, and levels of the faecal indicator organisms (FIOs) were closely associated with increased turbidity levels (presumptive E. coli and turbidity, r = 0.43). Spatio-temporal variation in microbial water quality parameters was accounted for by three principal components (67.6%). Cluster Analysis, reduced the fourteen monitoring sites to six representative ‘sentinel’ sites. The correlation coefficient between presumptive E. coli and phages of Bacteroides GB-124 was very small (r = 0.05) whilst that between turbidity and suspended solids was high (r = 0.62). Variations in climate, animal and anthropogenic interferences were all, either directly or indirectly, related to faecal contamination. The findings show the importance of meteorological conditions, such as storm events, on microbial water quality, and suggest that any future increases in the frequency of storm events (associated with climate change) are likely to result in a greater incidence of FIO/pathogen loads. This low-cost approach could help to predict spatio-temporal ‘hotspots’ of elevated waterborne disease risk. The work also represents an important step towards integrating novel MST tools into river catchment modelling.     

Contribution of treated wastewater to the microbiological quality of Seine River in Paris

Urban part of Seine River serving as drinking water supply in Paris can be heavily contaminated by Cryptosporidium spp. and Giardia duodenalis. In the absence of agricultural practice in this highly urbanized area, we investigated herein the contribution of treated wastewater to the microbiological quality of this river focusing on these two parasites. Other microorganisms such as faecal bacterial indicators, enteroviruses and oocysts of Toxoplasma gondii were assessed concurrently. Raw wastewaters were heavily contaminated by Cryptosporidium and Giardia (oo)cysts, whereas concentrations of both protozoa in treated wastewater were lower. Treated wastewater, flowed into Seine River, had a parasite concentration closed to the one found along the river, in particular at the entry of a drinking water plant (DWP). Even if faecal bacteria were reliable indicators of a reduction in parasite concentrations during the wastewater treatment, they were not correlated to protozoal contamination of wastewater and river water. Oocysts of T. gondii were not found in both raw and treated wastewater, or in Seine River. Parasitic contamination was shown to be constant in the Seine River up to 40 km upstream Paris. Altogether, these results strongly suggest that treated wastewater does not contribute to the main parasitic contamination of the Seine River usually observed in this urbanized area.     

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.     

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10² and 4.3 × 10² genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.     

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators

Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 10(4) copies/100 mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms.     

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.