The prevalence and proliferation of antibiotic resistant bacteria is profoundly important to human health, but the extent to which aquatic environments contribute toward the dissemination of ...antibiotic resistant genes (ARGs) is poorly understood. The prevalence of 24 ARGs active against eight antibiotic classes (β-lactams, aminoglycosides, glycopeptides, chloramphenicols, tetracycline, macrolides, trimethoprim, and sulfonamides) was evaluated in surface water samples collected from Germany and Australia with culture independent methods. The ARGs most frequently detected both in Germany and Australia were sulI, sulII (77–100%), and dfrA1 (43–55%) which code for resistance to sulfonamide and trimethoprim. Macrolides resistance gene ermB was relatively more prevalent in the surface water from Germany (68%) than Australia (18%). In contrast, the chloramphenicol resistance gene catII was more frequently detected in Australia (64%) than Germany (9%). Similarly, β-lactams resistance gene ampC was more prevalent in the samples from Australia (36%) than Germany (19%). This study highlights wide distribution of ARGs for sulfonamide, trimethoprim, macroline, β-lactams and chloramphenicol in the aquatic ecosystems. Aquatic ecosystems can therefore be reservoirs of ARGs genes which could potentially be transferred from commensal microorganisms to human pathogens.
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.
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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.
The concurrence of human sewage contamination in urban stormwater runoff (n=23) from six urban catchments across Australia was assessed by using both microbial source tracking (MST) and chemical ...source tracking (CST) markers. Out of 23 stormwater samples human adenovirus (HAv), human polyomavirus (HPv) and the sewage-associated markers; Methanobrevibacter smithii nifH and Bacteroides HF183 were detected in 91%, 56%, 43% and 96% of samples, respectively. Similarly, CST markers paracetamol (87%), salicylic acid (78%) acesulfame (96%) and caffeine (91%) were frequently detected. Twenty one samples (91%) were positive for six to eight sewage related MST and CST markers and remaining two samples were positive for five and four markers, respectively. A very good consensus (>91%) observed between the concurrence of the HF183, HAv, acesulfame and caffeine suggests good predictability of the presence of HAv in samples positive for one of the three markers. High prevalence of HAv (91%) also suggests that other enteric viruses may also be present in the stormwater samples which may pose significant health risks. This study underscores the benefits of employing a set of MST and CST markers which could include monitoring for HF183, adenovirus, caffeine and paracetamol to accurately detect human sewage contamination along with credible information on the presence of human enteric viruses, which could be used for more reliable public health risk assessments. Based on the results obtained in this study, it is recommended that some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.
•Presence of multiple MST and CST markers suggests ubiquitous sewage contamination.•MST and CST markers suggest ubiquitous sewage contamination in urban environment.•Good consensus (>80%) between the occurrence of MST and CST markers•HF183 had high concurrence with human adenovirus and acesulfame.
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.
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•Enteric virus removal efficacy of wastewater treatment plants with activated sludge (ASP) process was determined.•ASP in sub-tropical climate could be an effective treatment barrier with >3 log10 removal of enteric virus.•Adenovirus was more resistant to removal compared to polyomavirus and torque teno virus.•Physicochemical parameters of water quality are poor predictor of enteric virus presence.•Adenovirus could be used as a model microorganism for determining enteric virus removal efficacy.
Elevated numbers of enteric pathogens in the receiving waters following a storm event can be a serious public health concern. The purpose of this study was to conduct a preliminary investigation into ...the presence of human pathogens of concern in urban stormwater runoff. The involvement of a human sewage as a potential source of contamination was also investigated by using microbial source tracking methods. Water samples (20L) were collected after storm events and during the dry weather from six sites in Brisbane, Australia. Collected samples were analyzed for fecal indicator bacteria (FIB), and then concentrated using hollow fiber ultrafiltration followed by molecular detection of selected enteric pathogens. The levels of FIB were found to frequently exceed the upper limit of Australian guidelines for managing risks in recreational water, during the dry periods and by further several orders of magnitude in the stormwater runoff. Enterococcus spp. numbers as high as 3×104 100 mL−1 were detected in the stormwater runoff at the Fitzgibbon site. Human adenovirus and polyomavirus were frequently detected from all six sampling sites during wet and dry weather conditions suggesting their wide spread presence in the urban aquatic environments. Campylobacter jejuni, Campylobacter coli and Salmonella enterica were also detected during both dry and wet weather conditions. Presence of human-specific HF183 Bacteroides marker in most of the samples tested suggests ubiquitous sewage contamination in the urban environment. Since stormwater runoff routinely contains high numbers of FIB and other enteric pathogens, some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.
► FIB numbers in stormwater regularly exceeded water quality guidelines. ► Human adenovirus and polyomavirus were frequently detected in stormwater. ► C. jejuni, C. coli and Salmonella enterica were detected in both dry and wet periods. ► High prevalence of HF183 suggests ubiquitous sewage contamination in urban environment.
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.
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.
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