Prevalence of antibiotic resistance and virulence genes in the biofilms from an aquifer recharged with stormwater.

An improved understanding of the diversity and composition of microbial communities carrying antibiotic resistance genes (ARGs) and virulence genes (VGs) in aquifers recharged with stormwater is essential to comprehend potential human health risks from water reuse. A high-throughput functional gene array was used to study the prevalence of ARGs and VGs in aquifer biofilms (n = 27) taken from three boreholes over three months. Bacterial genera annotated as opportunistic pathogens such as Aeromonas, Burkholderia, Pseudomonas, Shewanella, and Vibrio were ubiquitous and abundant in all biofilms. Bacteria from clinically relevant genera, Campylobacter, Enterobacter, Klebsiella, Mycobacterium, Mycoplasma, and Salmonella were detected in biofilms. The mean travel time of stormwater from the injection well to P1 and P3 boreholes was 260 and 360 days respectively. The presence of ARGs and VGs in the biofilms from these boreholes suggest a high spatial movement of ARGs and VGs in the aquifer. The ARGs with the highest abundance were small multidrug resistance efflux pumps (SMR) and multidrug efflux (Mex) followed by ß-lactamase C genes. ß- lactamase C encoding genes were primarily detected in Enterobacteriaceae, Pseudomonadaceae, Bacillaceae, and Rhodobacteraceae families. The VGs encoding siderophores, including aerobactin (iro and iuc genes), followed by pilin, hemolysin, and type III secretion were ubiquitous. Canonical correspondence analysis suggested that Total Organic Carbon (TOC), Dissolved Organic Carbon (DOC), turbidity, and Fe concentration has a significant impact on the microbial community structure of bacteria carrying ARGs and VGs. Post abstraction treatment of groundwater may be prudent to improve water security and reduce potential health risks.

Decay rates of Escherichia coli, Enterococcus spp., F-specific bacteriophage MS2, somatic coliphage and human adenovirus in facultative pond sludge.

The purpose of this study was to evaluate the efficacy of a waste stabilization pond (WSP) system to reduce pathogen contaminants in sludge. This included examining the factors that influence the fate and concentration of human pathogens and their indicators in two sludge layers. The decay rates of five study microorganisms were determined under in-situ conditions at a WSP. The background levels of fecal origin microorganisms were consistently detected (ranging: Escherichia coli 104 to 106, enterococci 101 to 103, F-specific bacteriophage (MS2) 101 to 103 and somatic coliphage 101 to 104 colony-forming units (CFU) mL-1, as well as 101 to 102 human adenovirus gene copies mL-1) in the primary facultative pond. Among microorganisms tested, the bacteria generally decayed faster than adenovirus and bacteriophage, particularly in the upper sludge layer. Due to the observed regrowth of E. coli, it may have a limited value as an indicator for pathogen removal in the wastewater stabilization ponds. The abundance of E. coli numbers within the pond biome followed changes in pond temperature over time. The results of the study suggest that viruses could survive for a long time, particularly in deeper layers (>1 metre) in the sludge, during winter months (T90 = 156 d). The presence of human pathogens in WSP sludge, in particular viruses, may be a barrier to its beneficial reuse in agriculture. The results indicate that additional treatment of sludge may be required to mitigate potential public health risks from reuse of sludge for agricultural purposes.

Comparative removal of antibiotic resistance genes during chlorination, ozonation, and UV treatment.

Efficient treatment methods for the removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from drinking water are needed to reduce health risks. However, there is a lack of empirical data on ARB and ARG removal during conventional water disinfection processes. In this study, the removal of ARB and ARGs by three disinfection processes (chlorination, ozonation, and UV treatment) was investigated on a laboratory scale using Escherichia coli and Enterococcus faecium carrying ARGs. Bacterial inactivation was determined by plate count methods, and ARG damage was quantified using real-time PCR. Only for ozone treatment, similar inactivation rates for bacterial cells and ARGs were observed when 1 mg*L-1 of ozone, with a contact time of 5 min, was used, which resulted in a 5.0 log reduction of bacterial cells and a 4.3-4.6 log reduction of ARGs. For chlorine and UV, inactivation of bacterial cells was observed at lower doses than those needed for the decrease of ARG copy numbers. The use of 0.5 mg*L-1 free chlorine (30 min contact time) led to a 3.8-5.6 log reduction of the bacterial numbers and to a 0.8-2.8 log reduction of ARGs. Ultraviolet light irradiation with 600 J*m-2 resulted in a 4.8-5.5 log reduction of bacterial cells, but in a negligible reduction (0-1.0 log) of ARGs. Although UV and chlorine treatments were effective in the inactivation of bacterial cells, incomplete degradation of ARGs was observed. Therefore, plasmid-borne ARGs can potentially be transferred to other bacteria even after the disinfection process. Our results provide important insights into the fate of ARGs during drinking water disinfection processes.

Comparative enteric viruses and coliphage removal during wastewater treatment processes in a sub-tropical environment.

Microbiological safety of reclaimed water is one of the most important issues in managing potential health risks related to wastewater recycling. Presence and removal of human adenovirus (HAdV), human polyomavirus (HPyV), human torque teno virus (HTtV) and somatic coliphage family Microviridae in three wastewater treatment plants (WWTP) in sub-tropical Brisbane, Australia was investigated. All three WWTPs employ activated sludge process with added on Bardenpho process for nutrient removal. HPyV, HAdV, HTtV and Microviridae were consistently detected in the influent (105 to 106 Genomic copies (GC) L-1) and secondary treated effluent (102 to 103GCL-1). The results of this study suggest that, under appropriate conditions, WWTPs with activated sludge process in sub-tropical climate could be an effective treatment barrier with >3 log10 removal of enteric virus. The geometric mean of pooled data for each virus from all sites showed the highest removal for HPyV (3.65 log10) and lowest for HAdV (2.79 log10) which was statistically significant (p=0.00001). Whereas, the removal rate of HTtV and Microviridae was identical (2.81 log10). A poor correlation between the presence of enteric virus in influent or effluent with routinely monitored physicochemical parameters suggests limited use of physicochemical parameters as predictors of enteric virus presence. High prevalence of HAdV in influent and effluent combined with comparatively low removal suggest that it could be used as a model microorganism for determining enteric virus removal efficacy. Additional tertiary treatment may be required prior to effluent reuse for non-potable purposes or discharge into the recreational waters to prevent exposure of people to health hazards.

Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova.

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

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 blaTEM, blaSHV, 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.

Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks.

A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendall's tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data.

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods.

In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79-80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 10(5) ± 6.4 × 10(5) and 6.3 × 10(5) ± 4.7 × 10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 10(5) ± 1.5 × 10(4) (determined by qPCR) compared to 4.9 × 10(4) ± 3.7 × 10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

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(6) gene copies per ml), followed by those of HF183 (8.0 × 10(5) gene copies per ml) and Enterococcus spp. (3.6 × 10(5) 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.

Pathogen Decay during Managed Aquifer Recharge at Four Sites with Different Geochemical Characteristics and Recharge Water Sources.

Recycling of stormwater water and treated effluent via managed aquifer recharge (MAR) has often been hampered because of perceptions of low microbiological quality of recovered water and associated health risks. The goal of this study was to assess the removal of selected pathogens in four large-scale MAR schemes and to determine the influence of aquifer characteristics, geochemistry, and type of recharge water on the pathogen survival times. Bacterial pathogens tested in this study had the shortest one log removal time (, <3 d), followed by oocysts (, <120 d), with enteric viruses having the biggest variability in removal times (, 18 to >200 d). Human adenovirus and rotavirus were relatively persistent under anaerobic conditions (, >200 d). Human adenovirus survived longer than all the other enteric virus tested in the study and hence could be used as a conservative indicator for virus removal in groundwater during MAR. The results suggest that site-specific subsurface conditions such as groundwater chemistry can have considerable influence on the decay rates of enteric pathogens and that viruses are likely to be the critical pathogens from a public health perspective.

Global occurrence of Torque teno virus in water systems.

Bacterial indicator organisms are used globally to assess the microbiological safety of waters. However, waterborne viral outbreaks have occurred in drinking water systems despite negative bacterial results. Using viral markers may therefore provide more accurate health risk assessment data. In this study, fecal, wastewater, stormwater, surface water (fresh and salt), groundwater, and drinking water samples were analyzed for the presence or concentration of traditional indicators, innovative indicators and viral markers. Samples were obtained in the United States, Italy, and Australia and results compared to those reported for studies conducted in Asia and South America as well. Indicators included total coliforms, Escherichia coli, enterococci, male-specific coliphages, somatic coliphages and microviradae. Viral markers included adenovirus, polyomavirus, and a potential new surrogate, Torque teno virus (TTV). TTV was more frequently found in wastewaters (38-100%) and waters influenced by waste discharges (25%) than in surface waters used as drinking water sources (5%). TTV was also specific to human rather than animal feces. While TTV numbers were strongly correlated to other viral markers in wastewaters, suggesting its utility as a fecal contamination marker, data limitations and TTV presence in treated drinking waters demonstrates that additional research is needed on this potential viral indicator.

Comparison of concentration methods for rapid detection of hookworm ova in wastewater matrices using quantitative PCR.

Hookworm infection contributes around 700 million infections worldwide especially in developing nations due to increased use of wastewater for crop production. The effective recovery of hookworm ova from wastewater matrices is difficult due to their low concentrations and heterogeneous distribution. In this study, we compared the recovery rates of (i) four rapid hookworm ova concentration methods from municipal wastewater, and (ii) two concentration methods from sludge samples. Ancylostoma caninum ova were used as surrogate for human hookworm (Ancylostoma duodenale and Necator americanus). Known concentration of A. caninum hookworm ova were seeded into wastewater (treated and raw) and sludge samples collected from two wastewater treatment plants (WWTPs) in Brisbane and Perth, Australia. The A. caninum ova were concentrated from treated and raw wastewater samples using centrifugation (Method A), hollow fiber ultrafiltration (HFUF) (Method B), filtration (Method C) and flotation (Method D) methods. For sludge samples, flotation (Method E) and direct DNA extraction (Method F) methods were used. Among the four methods tested, filtration (Method C) method was able to recover higher concentrations of A. caninum ova consistently from treated wastewater (39-50%) and raw wastewater (7.1-12%) samples collected from both WWTPs. The remaining methods (Methods A, B and D) yielded variable recovery rate ranging from 0.2 to 40% for treated and raw wastewater samples. The recovery rates for sludge samples were poor (0.02-4.7), although, Method F (direct DNA extraction) provided 1-2 orders of magnitude higher recovery rate than Method E (flotation). Based on our results it can be concluded that the recovery rates of hookworm ova from wastewater matrices, especially sludge samples, can be poor and highly variable. Therefore, choice of concentration method is vital for the sensitive detection of hookworm ova in wastewater matrices.

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 (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

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.

Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium.

Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water.

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.

Relative inactivation of faecal indicator bacteria and sewage markers in freshwater and seawater microcosms.

UNLABELLED: In this study, the relative inactivation of faecal indicator bacteria (FIB) namely Escherichia coli, enterococci and sewage markers [Bacteroides HF183 and human adenoviruses (HAVs)] was assessed in sewage-spiked freshwater and seawater microcosms under ambient subtropical climatic conditions. The numbers of declining FIB were measured with culture-based methods, whereas the numbers of sewage markers were measured with qPCR assays. The T90 inactivation times of E. coli, enterococci and the HF183 markers in both freshwater and seawater microcosms were <3·5 days, suggesting the suitability of the HF183 marker to identify recent sewage pollution events. The T90 value of HAVs (9·4-13 days), however, was significantly higher than FIB and the HF183 marker in both freshwater (P < 0·001) and seawater (P < 0·05) microcosms. Therefore, we recommend that HAVs should be used as an additional marker to adequately assess the potential health risks associated with longer-term sewage-polluted environmental waters. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have shown that the persistence of the Bacteroides HF183 marker in freshwater and seawater microcosms was similar to faecal indicator bacteria (Escherichia coli and enterococci), whereas human adenoviruses (HAVs) persisted relatively longer. These findings suggest the suitability of both the markers to identify sewage pollution in environmental waters. However, HF183 marker appeared to be more useful than HAVs in identifying recent sewage pollution. As, HAVs may remain infective for lengthy periods, it should be used in conjunction with the HF183 marker to obtain information on the potential human health risks associated with sewage-polluted freshwater and seawater.

Decay of enteric microorganisms in biosolids-amended soil under wheat (Triticum aestivum) cultivation.

There is a growing need for better assessment of health risks associated with land-applied biosolids. This study investigated in-situ decay of seeded human adenovirus (HAdV), Salmonella enterica, Escherichia coli, and bacteriophage (MS2) in biosolids-amended soil under wheat cultivation. The biosolids seeded with microorganisms were placed in decay chambers which were then placed in the topsoil (10 cm depth) at three different sites. Sites were selected in arid wheat-growing regions of Australia with loamy-sand soil type (Western Australia) and sandy soil (South Australia). Seeded E. coli and S. enterica had a relatively short decay time (T90 = 4-56 days) in biosolids-amended soil compared to un-amended soil (T90 = 8-83 days). The decreasing soil moisture over the wheat-growing season significantly (P < 0.05) influenced survival time of both bacteria and MS2 at Western Australia (Moora) and South Australia (Mt Compass) sites, particularly in the un-amended soils. Increasing soil temperature also significantly (P < 0.05) influenced the decay of MS2 at these sites. In this study, no notable decline in HAdV numbers (PCR detectable units) was observed in both biosolids-amended and the un-amended soils at all three sites. The HAdV decay time (T90 ≥ 180 days) in biosolids-amended and un-amended soils was significantly higher than MS2 (T90 = 22-108 days). The results of this study suggest that adenovirus could survive for a longer period of time (>180 days) during the winter in biosolids-amended soil. The stability of adenovirus suggests that consideration towards biosolids amendment frequency, time, rates and appropriate withholding periods are necessary for risk mitigation.

Opportunistic pathogens in roof-captured rainwater samples, determined using quantitative PCR.

In this study, quantitative PCR (qPCR) was used for the detection of four opportunistic bacterial pathogens in water samples collected from 72 rainwater tanks in Southeast Queensland, Australia. Tank water samples were also tested for fecal indicator bacteria (Escherichia coli and Enterococcus spp.) using culture-based methods. Among the 72 tank water samples tested, 74% and 94% samples contained E. coli and Enterococcus spp., respectively, and the numbers of E. coli and Enterococcus spp. in tank water samples ranged from 0.3 to 3.7 log10 colony forming units (CFU) per 100 mL of water. In all, 29%, 15%, 13%, and 6% of tank water samples contained Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa and Legionella pneumophila, respectively. The genomic units (GU) of opportunistic pathogens in tank water samples ranged from 1.5 to 4.6 log10 GU per 100 mL of water. A significant correlation was found between E. coli and Enterococcus spp. numbers in pooled tank water samples data (Spearman's rs = 0.50; P < 0.001). In contrast, fecal indicator bacteria numbers did not correlate with the presence/absence of opportunistic pathogens tested in this study. Based on the results of this study, it would be prudent, to undertake a Quantitative Microbial Risk Assessment (QMRA) analysis of opportunistic pathogens to determine associated health risks for potable and nonpotable uses of tank water.

Prevalence of enterococcus species and their virulence genes in fresh water prior to and after storm events.

Enterococcus spp. isolates (n = 286) collected from six surface water bodies in subtropical Brisbane, Australia, prior to and after storm events, were identified to species level and tested for the presence of seven clinically important virulence genes (VGs). Enterococcus faecalis (48%), Enterococcus faecium (14%), Enterococcus mundtii (13%), and Enterococcus casseliflavus (13%) were frequently detected at all sites. The frequency of E. faecium occurrence increased from 6% in the dry period to 18% after the wet period. The endocarditis antigen (efaA), gelatinase (gelE), collagen-binding protein (ace), and aggregation substance (asa1) were detected in 61%, 43%, 43%, and 23% of Enterococcus isolates, respectively. The chances of occurrence of ace, gelE, efaA, and asa1 genes in E. faecalis were found to be much higher compared to the other Enterococcus spp. The observed odds ratio of occurrence of ace and gelE genes in E. faecalis was much higher at 7.96 and 6.40 times, respectively. The hyl gene was 3.84 times more likely to be detected in E. casseliflavus. The presence of multiple VGs in most of the E. faecalis isolates underscores the importance of E. faecalis as a reservoir of VGs in the fresh water aquatic environment. Consequently, if contaminated surface water is to be used for production of potable and nonpotable water some degree of treatment depending upon intended use such as detention in basins prior to use or chlorination is required.