Decay of human enteric pathogens in agricultural soil amended with biosolids: Key findings from a comprehensive research project to examine potential health risks.

A comprehensive study was undertaken to examine the survival potential of enteric microorganisms in biosolids-amended soil, wheat plant phyllosphere, and stored grains. The presence of these microorganisms in the dust at harvesting time was also evaluated. In situ field experiments were conducted to examine the decay of E. coli (indicator bacteria), Salmonella enterica, bacteriophage MS2 and human adenovirus in biosolids-amended soils and in dust generated during harvesting of wheat. Glasshouse experiments were conducted to determine the survival potential of enteric microorganisms in the wheat phyllosphere and stored grains to determine any possible risks to humans or livestock through consumption of contaminated grains or fodder. The results of this study suggest that the target microorganisms decayed faster in the biosolids-amended soil compared with the unamended soil in the field, that the decay times were specific to the microorganism type; and that microorganism decay was correlated to declining soil moisture levels and increasing soil temperature. The risk of transmission of disease-causing microorganisms (human pathogens) from cereal crops fertilised with biosolids was considered to be low

Decay of Escherichia Coli in Biosolids Applied to Agricultural Soil

There is little scientific data available on the survival patterns of pathogenic microorganisms introduced into the soil through the broad acre application of biosolids. This study was conducted to investigate the decay rates of Escherichia coli in agricultural soil amended with biosolids during two different growing seasons in a dry temperature cropping region in Western Australia.Biosolids-amended and unamended soil were inoculated with E. coli (ACM 1803), inserted into sentinel chambers and placed into the topsoil (0-10 cm) of a wheat crop. Biosolids were applied to designated biosolids plots, according to normal district practice, and E. coli numbers within the sentinel chambers were monitored over time. E. coli numbers in biosolids-amended soil reached detection limits (>10 cfu/mL) within 6 to 7 months. The decay patterns of E. coli, by treatment difference (biosolids-amended or unamended), linear and quadratic relationships of sampling time, and their interactions were highly significant. The T90 or time taken for a 90% reduction in numbers in the biosolids-amended soil was estimated to be 74, 143, 183 days (2006) and 173, 211 days (2008) as compared with 188 days (2006) and 156, 242 days (2008) in the unamended soil. This research provides scientific data on the survival times of E. coli in agricultural soil, with and without biosolids and can thus be helpful to public health policy

Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ∼10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg(2+)) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters

Distributions of fecal markers in wastewater from different climatic zones for human fecal pollution tracking in Australian surface waters

Recreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp., HFMs Bacteroides HF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia. E. coli mean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 10 gene copies per ml), followed by those of HF183 (8.0 × 10 gene copies per ml) and Enterococcus spp. (3.6 × 10 gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated that Bacteroides HF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters

Toolbox approaches using molecular markers and 16S rRNA gene amplicon data sets for identification of fecal pollution in surface water

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small

Antibiotic resistance and virulence genes in coliform water isolates

Widespread fecal pollution of surface water may present a major health risk and a significant pathway for dissemination of antibiotic resistance bacteria. The River Rhine is one of the longest and most important rivers in Europe and an important raw water source for drinking water production. A total of 100 coliform isolates obtained from River Rhine (Germany) were examined for their susceptibility to seven antimicrobial agents. Resistances against amoxicillin, trimethoprim/sulfamethoxazole and tetracycline were detected in 48%, 11% and 9% of isolates respectively. The antibiotic resistance could be traced back to the resistance genes bla, bla, ampC, sul1, sul2, dfrA1, tet(A) and tet(B). Whereby, the ampC gene represents a special case, because its presence is not inevitably linked to a phenotypic antibiotic resistance. Multiple antibiotics resistance was often accompanied by the occurrence of class 1 or 2 integrons. E. coli isolates belonging to phylogenetic groups A and B1 (commensal) were more predominant (57%) compared to B2 and D groups (43%) which are known to carry virulent genes. Additionally, six E. coli virulence genes were also detected. However, the prevalence of virulence genes in the E. coli isolates was low (not exceeding 4.3% per gene) and no diarrheagenic E. coli pathotypes were detected. This study demonstrates that surface water is an important reservoir of ARGs for a number of antibiotic classes such as sulfonamide, trimethoprim, beta-lactam-antibiotics and tetracycline. The occurrence of antibiotic resistance in coliform bacteria isolated from River Rhine provides evidence for the need to develop management strategies to limit the spread of antibiotic resistant bacteria in aquatic environment

Behaviour and fate of nine recycled water trace organics during managed aquifer recharge in an aerobic aquifer

The fate of nine trace organic compounds was evaluated during a 12 month large-scale laboratory column experiment. The columns were packed with aquifer sediment and evaluated under natural aerobic and artificial anaerobic geochemical conditions, to assess the potential for natural attenuation of these compounds during aquifer passage associated with managed aquifer recharge (MAR). The nine trace organic compounds were bisphenol A (BPA), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMOR), carbamazepine, oxazepam, iohexol and iodipamide. In the loworganic carbon content Spearwood sediment, all trace organicswere non-retardedwith retardation coefficients between 1.0 and 1.2, indicating that these compounds would travel at near groundwater velocities within the aquifer. The natural aerobic geochemical conditions provided a suitable environment for the rapid degradation for BPA, E2, iohexol (half life b1 day). Lag-times for the start of degradation of these compounds ranged from b15 to 30 days. While iodipamide was persistent under aerobic conditions, artificial reductive geochemical conditions promoted via the addition of ethanol, resulted in rapid degradation (half life b1 days). Pharmaceuticals (carbamazepine and oxazepam) and disinfection by-products (NDMA and NMOR) did not degrade under either aerobic or anaerobic aquifer geochemical conditions (half life N50 days). Field-based validation experiments with carbamazepine and oxazepam also showed no degradation. If persistent trace organics are present in recycled waters at concentrations in excess of their intended use, natural attenuation during aquifer passage alonemay not result in extracted watermeeting regulatory requirements. Additional pre treatment of the recycled water would therefore be required

Seasonal Abundance of Fecal Indicators and Opportunistic Pathogens in Roof-Harvested Rainwater Tanks

Here we provide seasonal data on the concentrations of total coliform, 'Escherichia coli' and 'Enterococcus' spp. and six opportunistic pathogens ('Acanthamoeba' spp., 'Legionella' spp., 'Legionella pneumophila', 'Mycobacterium avium', 'Mycobacterium intracellulare', and 'Pseudomonas aeruginosa') of public health significance in 24 tank water samples over six monthly sampling events from August 2015 to March 2006. Quantitative PCR (qPCR) assays were chosen for the quantification of six opportunistic pathogens and culture-based methods were used for the enumeration of fecal indicators. The data fle has been stored in a publicly available repository. The data on concentrations of opportunistic pathogens in RHRW will provide information for rainwater users regarding potential seasonality of risks. Quantitative data presented in this study can be used to perform quantitative microbial risk assessment (QMRA) of RHRW for various potable and nonpotable uses. Data can be used by health regulators to develop guidelines related to RHRW. Funding statement: This research was undertaken and funded as part of a Fulbright-CSIRO Postgraduate Scholarship sponsored by the CSIRO Land and Water Flagship

Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method

The risk of human hookworm infections from land application of wastewater matrices could be high in regions with high hookworm prevalence. A rapid, sensitive and specific hookworm detection method from wastewater matrices is required in order to assess human health risks. Currently available methods used to identify hookworm ova to the species level are time consuming and lack accuracy. In this study, a real-time PCR method was developed for the rapid, sensitive and specific detection of canine hookworm (Ancylostoma caninum) ova from wastewater matrices. A. caninum was chosen because of its morphological similarity to the human hookworm (Ancylostoma duodenale and Necator americanus). The newly developed PCR method has high detection sensitivity with the ability to detect less than one A. caninum ova from 1 L of secondary treated wastewater at the mean threshold cycle (CT) values ranging from 30.1 to 34.3. The method is also able to detect four A. caninum ova from 1 L of raw wastewater and from ~4 g of treated sludge with mean CT values ranging from 35.6 to 39.8 and 39.8 to 39.9, respectively. The better detection sensitivity obtained for secondary treated wastewater compared to raw wastewater and sludge samples could be attributed to sample turbidity. The proposed method appears to be rapid, sensitive and specific compared to traditional methods and has potential to aid in the public health risk assessment associated with land application of wastewater matrices. Furthermore, the method can be adapted to detect other helminth ova of interest from wastewater matrices