Article

Unraveling the riverine antibiotic resistome: The downstream fate of anthropogenic inputs

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Abstract

River networks are one of the main routes by which the public could be exposed to environmental sources of antibiotic resistance, that may be introduced e.g. via treated wastewater. In this study, we applied a comprehensive integrated analysis encompassing mass-flow concepts, chemistry, bacterial plate counts, resistance gene quantification and shotgun metagenomics to track the fate of the resistome (collective antibiotic resistance genes (ARGs) in a microbial community) of treated wastewater in two Swiss rivers at the kilometer scale. The levels of certain ARGs and the class 1 integron integrase gene (intI1) commonly associated with anthropogenic sources of ARGs decreased quickly over short distances (2-2.5 km) downstream of wastewater discharge points. Mass-flow analysis based on conservative tracers suggested this decrease was attributable mainly to dilution but ARG loadings frequently also decreased (e.g., 55.0-98.5 % for ermB and tetW) over the longest studied distances (6.8 and 13.7 km downstream). Metagenomic analysis confirmed that ARG of wastewater-origin did not persist in rivers after 5 ∼ 6.8 km downstream distance. sul1 and intI1 levels and loadings were more variable and even increased sharply at 5 ∼ 6.8 km downstream distance on one occasion. While input from agriculture and in-situ positive selection pressure for organisms carrying ARGs cannot be excluded, in-system growth of biomass is a more probable explanation. The potential for direct human exposure to the resistome of wastewater-origin thus appeared to typically abate rapidly in the studied rivers. However, the riverine aquatic resistome was also dynamic, as evidenced by the increase of certain gene markers downstream, without obvious sources of anthropogenic contamination. This study provides new insight into drivers of riverine resistomes and pinpoints key monitoring targets indicative of where human sources and exposures are likely to be most acute.

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... Wastewater treatment plants (WWTPs) are a known route through which sewage-borne resistance genes are discharged into the environment (Bürgmann et al., 2018;Rizzo et al., 2013). Even though many studies showed that the level (genes per volume, or absolute abundance) of ARGs decreases during wastewater treatment processes (Ju et al., 2019;Marano et al., 2020;Rodriguez-Mozaz et al., 2015), profound levels of ARGs remain in treated effluents (Lee et al., 2021;Rodriguez-Mozaz et al., 2015). For instance, the levels of common ARGs and bacteria in treated effluents are significantly higher than upstream waters by up to 2 order of magnitude (Lee et al., 2021;Rodriguez-Mozaz et al., 2015;Sabri et al., 2018). ...
... Even though many studies showed that the level (genes per volume, or absolute abundance) of ARGs decreases during wastewater treatment processes (Ju et al., 2019;Marano et al., 2020;Rodriguez-Mozaz et al., 2015), profound levels of ARGs remain in treated effluents (Lee et al., 2021;Rodriguez-Mozaz et al., 2015). For instance, the levels of common ARGs and bacteria in treated effluents are significantly higher than upstream waters by up to 2 order of magnitude (Lee et al., 2021;Rodriguez-Mozaz et al., 2015;Sabri et al., 2018). Accordingly, the levels of ARGs and bacteria at receiving waters increase after receiving effluents (Lee et al., 2021;Rodriguez-Mozaz et al., 2015), even though the exact degree of increase depends on the proportion of effluent discharge to river discharge (Lee et al., 2021;Ort and Siegrist, 2009). ...
... For instance, the levels of common ARGs and bacteria in treated effluents are significantly higher than upstream waters by up to 2 order of magnitude (Lee et al., 2021;Rodriguez-Mozaz et al., 2015;Sabri et al., 2018). Accordingly, the levels of ARGs and bacteria at receiving waters increase after receiving effluents (Lee et al., 2021;Rodriguez-Mozaz et al., 2015), even though the exact degree of increase depends on the proportion of effluent discharge to river discharge (Lee et al., 2021;Ort and Siegrist, 2009). ...
Article
Untreated combined sewage (bypass) is often discharged by wastewater treatment plants to receiving rivers during stormwater events, where it may contribute to increased levels of antibiotic resistance genes (ARGs) and multi-resistance risk factors (multi-resistant bacteria and multi-resistance genomic determinants (MGDs)) in the receiving water. Other contamination sources, such as soil runoff and resuspended river sediment could also play a role during stormwater events. Here we report on stormwater event-based sampling campaigns to determine temporal dynamics of ARGs and multi-resistance risk factors in bypass, treated effluent, and the receiving river, as well as complimentary data on catchment soils and surface sediments. Both indicator ARGs (qPCR) and resistome (ARG profiles revealed by metagenomics) indicated bypass as the main contributor to the increased levels of ARGs in the river during stormwater events. Furthermore, we showed for the first time that the risk of exposure to bypass-borne multi-resistance risk factors increase under stormwater events and that many of these MGDs were plasmid associated and thus potentially mobile. In addition, elevated resistance risk factors persisted for some time (up to 22 hours) in the receiving water after stormwater events, likely due to inputs from distributed overflows in the catchment. This indicates temporal dynamics should be considered when interpreting the risks of exposure to resistance from event-based contamination. We propose that reducing bypass from wastewater treatment plants may be an important intervention option for reducing dissemination of antibiotic resistance.
... In fact, only two CRE isolates were recovered from site P6, located approximately 5 Km downstream the WWTP-1, in 2019 and, at the same time, bla KPC abundance decreased approximately one order of magnitude between sites P3 and P6 in 2018. This dilution effect has already been described, with levels of ARGs decreasing rapidly over 2.0-2.5 Km distance from the effluent discharge into the river (Lee et al., 2021). The clonal relatedness between isolates obtained in different sampling campaigns and sampling sites suggests either persistence along the river and over time or the existence of a common source responsible for the continuous discharge of these isolates into the Lis River ( Figure S3). ...
... Other studies also report a similar trend, with tetracycline resistance genes being amongst the most abundant ARGs in rivers (Cacace et al., 2019;Tuo et al., 2018). Nonetheless, tetA gene concentrations detected here were one to four orders of magnitude higher than the concentrations reported in European WWTPs (Cacace et al., 2019;Lekunberri et al., 2017;Paulus et al., 2020Paulus et al., , 2019Reichert et al., 2021) and up to 5 orders of magnitude higher than the concentrations in some rivers located in Europe and Asia (Berglund et al., 2015;Lee et al., 2021;Rodriguez-Mozaz et al., 2015;Tuo et al., 2018). ...
... However, anthropogenic influences on ARGs profiles in waterways may change with the scale of the city through which the river flows. Although the abundance of ARGs is known to significantly decrease over short distances through dilution effects (Lee et al., 2021), the abundance of resistance genes in rivers may alter through the influence of urban conurbations located along the river. However, the dynamics and underlying mechanisms of ARGs under multi-urban influences along rivers have not been well studied. ...
... For example, in this study the absolute resistome abundance decreased at H3, H5, H8 and H16, perhaps due to hydrological effects, for example, a dilution effect as a direct consequence of a river confluence adjacent to each of the sites. Such a dilution effect is known to strongly affect resistome profiles over short distances (Lee et al., 2021). Although no significant differences were found between either N2 vs H10 or N4 vs H16, perhaps due to the unexpected input of antibiotic resistome contaminated effluent (e.g., sewage discharge without due approval), in general the resistome decreased with increasing distance from urban areas (P = 0.017) (Fig. S2). ...
Article
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With growing concerns about antibiotic resistance, the tracking of antibiotic resistance genes (ARGs) in urban waterways will facilitate our increased understanding of the impact of urbanization on ARGs dissemination. In the current study, we assessed the ARGs profiles and antibiotic resistome in water samples along the Jiulong River basin, a distance of 250 km, to better understand the impact of anthropogenic activities. A total of 244 ARGs and 12 MGEs were detected from 21 sampling sites. Both relative and absolute abundance of the observed resistome decreased with increasing distance from urban areas. Ordinary least-squares (OLS) regression revealed that both the relative and absolute resistome abundance were positively correlated with city size. The resistome had several inputs and outputs and Fast Expectation Maximization Microbial Source Tracking (FEAST), suggested that the majority of the antibiotic resistome originated from anthropogenic activities. A total of 8 ARGs and 20 microbial OTUs were considered as biomarkers that differentiated the location of sampling sites. Bacterial communities were significantly correlated with ARGs according to Procrustes analysis and Mantel test, which was also supported by a co-occurrence network. Variation partitioning analysis revealed that ARG profiles were driven by multiple factors. Although antibiotic resistome abundance significantly increased near urban conurbations, overall resistome abundance decreased as the river flowed downstream. Our study highlights the effect of conurbation size on antibiotic resistance profiles within the river basin and the potential resilience of rivers to recover from ARGs contamination.
... In fact, only two CRE isolates were recovered from site P6, located approximately 5 Km downstream the WWTP-1, in 2019 and, at the same time, bla KPC abundance decreased approximately one order of magnitude between sites P3 and P6 in 2018. This dilution effect has already been described, with levels of ARGs decreasing rapidly over 2.0-2.5 Km distance from the effluent discharge into the river (Lee et al., 2021). The clonal relatedness between isolates obtained in different sampling campaigns and sampling sites suggests either persistence along the river and over time or the existence of a common source responsible for the continuous discharge of these isolates into the Lis River ( Figure S3). ...
... Other studies also report a similar trend, with tetracycline resistance genes being amongst the most abundant ARGs in rivers (Cacace et al., 2019;Tuo et al., 2018). Nonetheless, tetA gene concentrations detected here were one to four orders of magnitude higher than the concentrations reported in European WWTPs (Cacace et al., 2019;Lekunberri et al., 2017;Paulus et al., 2020Paulus et al., , 2019Reichert et al., 2021) and up to 5 orders of magnitude higher than the concentrations in some rivers located in Europe and Asia (Berglund et al., 2015;Lee et al., 2021;Rodriguez-Mozaz et al., 2015;Tuo et al., 2018). ...
Article
We determined the distribution and temporal variation of Carbapenem Resistant Enterobacterales (CRE), carbapenemase-encoding genes and other antibiotic resistance genes (ARGs) in a highly polluted river (Lis River; Portugal), also assessing the potential influence of water quality to this distribution. Water samples were collected in two sampling campaigns performed one year apart (2018/2019) from fifteen sites and water quality was analyzed. CRE were isolated and characterized. The abundance of four ARGs (blaNDM, blaKPC, tetA, blaCTX-M), two Microbial Source Tracking (MST) indicators (HF183 and Pig-2-Bac) and the class 1 integrase gene (IntI1) was measured by qPCR. Results confirmed the poor quality of the Lis River water, particularly in sites near pig farms. A collection of 23 CRE was obtained: Klebsiella (n = 19), Enterobacter (n = 2) and Raoultella (n = 2). PFGE analysis revealed a clonal relationship between isolates obtained in different sampling years and sites. All CRE isolates exhibited multidrug resistance profiles. Klebsiella and Raoultella isolates carried blaKPC while Enterobacter harbored blaNDM. Conjugation experiments were successful for only four Klebsiella isolates. All ARGs were detected by qPCR on both sampling campaigns. An increase in ARGs and IntI1 abundances was detected in sites located downstream of wastewater treatment plants. Strong correlations were observed between blaCTX-M, IntI1 and the human-pollution marker HF183, and also between tetA and the pig-pollution marker Pig-2-bac, suggesting that both human- and animal-derived pollution in the Lis River are a potential source of ARGs. Plus, water quality parameters related to eutrophication and land use were significantly correlated with ARGs abundances. Our findings demonstrated that the Lis River encloses high levels of antibiotic resistant bacteria and ARGs, including CRE and carbapenemase-encoding genes. Overall, this study provides a better understanding on the impacts of water pollution resulting from human and animal activities on the resistome of natural aquatic systems.
... In turn, the resistome of biofilms collected at the WWTP inlet was enriched in genes encoding resistance to aminoglycosides, bacitracin, β-lactams, multidrug and sulfonamides, suggesting that the WWTP acts as a sink of ARGs circulating among the served population (Fig. 4). It is also important to highlight that the resistome profile of biofilms collected at the WWTP is similar to those previously described from raw sewage and, remarkably, from surface waters receiving WWTP effluents [81][82][83]. Among all other genes, the prevalence of sul1 also agrees with previous studies carried out in a raising main sewer pipe [2] and confirms its widespread distribution among sewage bacteria [30,61,82]. ...
... Particularly, the WWTP inlet was characterized by a high abundance of ISCR8, which is associated with genes encoding degradative enzymes involved in the catabolism of monocyclic nitroaromatic compounds [84,85], and intI1, which encodes the integrase of Class I integrons and proposed as proxy for anthropogenic pollution [37]. Given that intl1 is usually strongly correlated with high abundance of ARGs (e.g., sul1 and tetG, among others), its abundance is considered a good measure of general selective pressure and it has been detected in both water and stream biofilms collected in surface waterbodies receiving wastewater discharges [81,83,86]. Overall, the mobilome profile of the biofilm at the inlet of the WWTP suggested a great contribution of MGEs to the acquisition and mobilization of ARGs in the system. ...
Article
Oxidative chemicals, such as nitrate, are periodically added to sewer systems to mitigate sulfide production and its accumulation but data are lacking on how these treatments affect sewage microbiota and alter their gene expression and mobilization. The present study investigated such effects on the biofilm of a full-scale sewer collected at two different locations, namely a pumping station and at the inlet of an urban wastewater treatment plant (WWTP), before and 15 days after nitrate dosage using a combination of culture-dependent and -independent approaches. Nitrate dosing resulted ineffective on the concentration of antibiotic-resistant Escherichia coli (AR-EC) in biofilms but greatly affected the composition of biofilm bacterial communities and the associated resistome and mobilome, especially at the pumping station where nitrate was dosed. Such responses consisted of a clear reduction on strict anaerobes; an almost twofold increase on the expression of recA gene (stress-response marker); and a significant increase on the relative abundance of most antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In turn, the effects of nitrate dosing at the WWTP inlet were barely visible, suggesting that they vanished into the distance from the pumping site (2.4 km). Remarkably, however, the relative abundance of both resistance and mobilization gene biomarkers at the inlet of the studied WWTP clearly oversized that at the pumping station confirming that these facilities are sinks where these resistance determinants accumulate and propagate. Considering the abundance of resistant bacteria and genes in urban sewage, the effects of dosing chemicals should be carefully assessed to lessen the load of such biological pollutants into WWTPs.
... South-north differences in ARG occurrence and types were observed in this study, except for surface bulk soils. Different environmental parameters, climatic conditions, and the extent of human activities seem to be responsible for differences in ARG occurrence [53,54]. The Qinling-Huaihe Line can be regarded as an important geographical division between northern and southern China. ...
Article
Full-text available
Background Riverine ecosystems are one of the most important reservoirs of antibiotic resistance genes (ARGs) in the environment, but the occurrence and controlling factors of ARG distribution in different habitats of riverine ecosystems remain poorly understood. In this study, a metagenomic approach was used to characterize ARG types and their abundance in different habitats (rhizosphere soil, surface bulk soil, bottom bulk soil, and sediment) of riverine ecosystems in eastern China. Sampling sites were located along different rivers of eastern China, which are geographically isolated. Differences in bacterial communities, mobile genetic elements (MGEs), pattern and intensity of human activities, climate, and other environmental factors at the sampling sites and habitats were expected to affect ARG occurrence. Results ARGs were observed with high variations in diversity (44–206 subtypes) and abundance (6.85–105.68 ×/Gb). There were significant south-north differences in ARG occurrence in the same habitat, except for surface bulk soil. And the significant difference was found in ARGs among four southern habitats. South–north differences in ARGs of the same habitat were mainly attributed to the combination of different occurrence frequencies and habitat selections of ARGs. Differences in ARG profiles among the four habitats in the south and the north were both mainly attributed to the different occurrence frequencies of ARGs. Bacterial communities and MGEs (Mobile genetic elements) could account for the observed variance in the resistome of riverine ecosystems across eastern China. The co-occurrences of specific ARGs with bacterial communities and MGEs were more frequent at the northern sampling sites than in the south, and co-occurrence patterns (i.e. ARGs and bacterial communities or ARGs and MGEs) varied between the habitats. Moreover, building land in all habitats, except bulk soils, showed significant positive correlations with ARG abundance. Conclusion This study reveals a high variance in the resistome of riverine ecosystems in eastern China and its controlling factors. We appeal to the importance of assessment of ARGs in the riverine ecosystem and the need for future prevention and intervention of ARG spread.
... With the advent of next generation DNA sequencing, high microbial diversity has been revealed in various natural systems (Bardgett and van der Putten, 2014;Donaldson et al., 2015;Fuhrman et al., 2015), including stream biofilms (Battin et al., 2016;Nega et al., 2019;Peng et al., 2018). The challenge remains to establish the determinants of microbial diversity patterns in nature and how microbial communities may respond to environmental perturbations (Lee et al., 2021;Mansfeldt et al., 2020). Here we studied microbial diversity patterns of stream biofilms in four Swiss lowland streams and tested how bacterial and eukaryotic communities respond to wastewater, which contains microorganisms, micropollutants (MPs) and nutrients. ...
Article
Full-text available
Microbial life in natural biofilms is dominated by prokaryotes and microscopic eukaryotes living in dense association. In stream ecosystems, microbial biofilms influence primary production, elemental cycles, food web interactions as well as water quality. Understanding how biofilm communities respond to anthropogenic impacts, such as wastewater treatment plant (WWTP) effluent, is important given the key role of biofilms in stream ecosystem function. Here, we implemented 16S and 18S rRNA gene sequencing of stream biofilms upstream (US) and downstream (DS) of WWTP effluents in four Swiss streams to test how bacterial and eukaryotic communities respond to wastewater constituents. Stream biofilm composition was strongly affected by geographic location – particularly for bacteria. However, the abundance of certain microbial community members was related to micropollutants in the wastewater – among bacteria, micropollutant-associated members were found e.g. in Alphaproteobacteria, and among eukaryotes e.g. in Bacillariophyta (algal diatoms). This study corroborates several previously characterized responses (e.g. as seen in diatoms), but also reveals previously unknown community responses – such as seen in Alphaproteobacteria. This study advances our understanding of the ecological impact of the current wastewater treatment practices and provides information about potential new marker organisms to assess ecological change in stream biofilms.
... As expected, the tributaries were the overwhelming contribution of the dispersal source to the Chishui River, and the other external immigrations may have a limited dispersal to the river. This was inconsistent with the previous studies, which showed that livestock, domestic sewage discharge, and agriculture were the main contributors of river pollution (Liu G. et al., 2018;Lee et al., 2021). This discrepancy might be explained by the Chishui River is located in a karst area, and the external immigrations cannot reach the Chishui River due to that the immigrations may infiltrate into the groundwater quickly. ...
Article
Full-text available
Karst rivers are particularly vulnerable to bacterial pollution because immigrations are easily diffused from the surrounding environments due to their strong hydraulic connectivity. However, the assembly mechanism in shaping riverine bacterial biogeography is still poorly understood, especially for an ecosystem in the karst area. Here, 16S rRNA genes were used to explore the spatiotemporal and biogeographical patterns of bacterial communities from the Chishui River in the dry and wet seasons, and explore the impact of external immigration on the assembly of water bacterial communities. Our results showed clear spatiotemporal patterns of bacterial communities with a more pronounced seasonal rather than spatial fluctuation, which appeared to be dependent on seasonal-related environmental factors (e.g., temperature and turbidity). The bacterial communities exhibited a significant ( p < 0.05) distance–decay pattern in both seasons, and they had a stronger distance–decay relationship in the dry season than in the wet season. However, most of the biomarkers of different external immigrations did not show significant ( p > 0.05) distance–decay patterns along the Chishui river, implying that the biomarkers could be used as indicators of external immigration (e.g., OTU_125 and OTU_536). Also, the tributaries were the main external immigration (20.44–83.68%) for the Chishui River, while other terrestrial immigration (e.g., livestock, the soil of the cropland, brewing wastewater treatment plant, and sewages) showed relatively little influence, which could be due to the hydrodynamic conditions (e.g., fragile rock–soil system and hydrological structure) of the karst river. Additionally, the assembly of water bacterial communities in the Chishui river was governed by more determinism (50.7–85.7%) than stochasticity (14.3–49.3%) in both the dry and wet seasons. We demonstrated that the bacterial community’s substantial variations are largely shaped by deterministic processes, thereby providing a better understanding of spatiotemporal patterns and mechanisms of the bacterial community in karst river waters.
... This is consistent with our study that river inputs bring antibiotic pollution and affect downstream antibiotic concentration. Furthermore, Antibiotics from river input would influence the ecological risk of the receiving lake and increase the uncertainty about the risk of microbial resistance (Lee et al., 2021;Zhang et al., 2020a). Existing studies have shown that river input areas had the highest total antibiotics with >400 ng/g in sediment and 1500 ng/L in water , which was higher than that in the IW subarea (<400 ng/L in water and 120 ng/g in sediments) the current study. ...
Article
Antibiotics are widely used and ubiquitous in the environment, which in turn poses potential threat to human health. However, the effects of agricultural activities and river input on the fate and ecological risks of antibiotics in shallow lake are still poorly understood. Surface water, overlying water and pore water, sediments and aquatic plant samples in the historical planting subarea (PA), historical aquaculture subarea (AU), inflow subarea (IW), discharge subarea (DC), and conservation subarea (CK) of Baiyangdian Lake were collected and analyzed. Our results revealed that the total antibiotic concentrations ranged from 85.33 ng/L to 1631.47 ng/L in waters and from 66.90 ng/g to 177.03 ng/g in sediments. Generally, the total antibiotic concentrations introduced by planting activity in surface water, overlying water and sediments were higher and the levels of total antibiotics in pore water were more affected by river input. In addition, three quinolones (QNs) and two tetracyclines (TCs) were dominant antibiotics in almost five subareas. The pseudo-partitioning coefficient kd(pw) and bioaccumulation factor (BAF) of antibiotics varied according to the effects of river input and historical agricultural activities. The ecological risk (RQ) of antibiotics from agricultural activities was higher than that from river input. The norfloxacin (NOR) in pore water showed high RQ, which contributed to a large proportion (>50 %) of the combined ecological risks (∑RQs) except for surface water. Therefore, NOR should be used as the primary ecological risk control index for antibiotic contamination management in the BYD. ∑RQs showed high risk in water in the five subareas. This study can act as a reference for governments to formulate effective management strategies for protecting the ecological health of lakes.
... There were 33 ARG subtypes detected across microbiomes of all the ecosystem categories and carried by a wide lineage of bacterial species (Fig. S4 and Table S2). For example, the homologues of bacA (also known as UppP, undecaprenyl pyrophosphate phosphatases) were present in 2712 MAGs and 531 OTUs, which showed higher natural prevalence in the environmental bacteria (Van Goethem et al., 2018;Lee et al., 2021). Other genes mostly belong to efflux/transporter systems, such as multidrug-transporter, acrB, mdtK, mdtB, and tolC. ...
Article
Widespread antibiotic resistance across Earth's habitats has become a critical health concern. However, large-scale investigation on the distribution of antibiotic resistance genes (ARGs) in the microbiomes from most types of ecosystem is still lacking. In this study, we provide a comprehensive characterization of ARGs for 52,515 microbial genomes covering various Earth's ecosystems, and conduct the risk assessment for ARG-carrying species based on further identification of mobile genetic elements (MGEs) and virulence factor genes (VFGs). We identify a total of 6159 ARG-carrying metagenome-assembled genomes (ACMs), and most of them are recovered from human gut and city subway. Our results show that efflux pump is the most common mechanism for bacteria to acquire multidrug resistance genes in Earth's microbiomes. Enterobacteriaceae species are the largest hosts of ARGs, accounting for 14% of total ACMs with 64% of the total ARG hits. Most of ARG-carrying species are unique in the different ecosystem categories, while 33 potential background ARGs are commonly shared by all ecosystem categories. We then detect 36 high-risk ARGs that likely threat public health in all ACMs. Based on ranking the importance of ARG-carrying species in the different ecosystem categories, several bacterial taxa such as Escherichia coli, Enterococcus faecalis, and Pseudomonas_A stutzeri were recognized as priority species for surveillance and control. Overall, our study gives a broad view of ARG-host associations in the environments.
... Although tetO exhibited the highest human health hazard score within the module, its prevalence in agricultural environments makes the ARG a lower priority as a wastewater indicator (Eramo et al., 2020;Freeman et al., 2018). ermB was also ranked highly as a hazard to human health and very common ARG in wastewater (Bengtsson-Palme et al., 2019;Lee et al., 2021;Sabri et al., 2020). Thus, the multidrug ARG ermB is a particularly strong candidate for monitoring resistance risk in wastewater for module-2. ...
Article
Antibiotic resistance is one of the greatest threats to global human and animal health of our time. Municipal wastewater has been identified as a hotspot of antibiotic resistance contamination to water bodies. However, there are numerous potential antibiotic resistant pathogens and their associated antibiotic resistance genes (ARGs), making it difficult to implement routine monitoring that addresses the breadth of the problem. The objective of this study was to identify candidate indicator ARGs for monitoring antibiotic resistance in wastewater and receiving water bodies. We developed a framework to identify indicator ARGs that incorporates clinical relevance, abundance in wastewater, geographic ubiquity, environmental relevance, ARG mobility, associations with mobile genetic elements, and the availability of quantitative analytical methods. To identify indicator ARGs, published metagenomic sequencing data from 191 wastewater samples originating from 64 countries across the world were obtained from online public repositories. Through ARG annotation and network analysis, this framework revealed 56 candidate indicator ARGs distributed across four modules of strongly correlated ARGs, with one ARG from each module (oqxA, ermB, sul1, and mexE) proposed as a minimally redundant monitoring target. The results of this study provide the basis for antibiotic resistance surveillance and monitoring framework in wastewater and contaminated waterways.
... With rapid urbanization and industrialization, the river is a crucial reservoir for ARGs and ARPBs in many countries (Böger et al., 2021;Proia et al., 2018;Wu et al., 2019). Moreover, river is an important environmental source of human exposure to antibiotics resistance (Lee et al., 2021). Thus, increasing studies have focused on ARGs and ARPBs in urban rivers in the last decade (Chen and Zhou, 2014;Dang et al., 2017;Elder et al., 2021). ...
Article
Rivers are important environmental sources of human exposure to antibiotic resistance. Many factors can change antibiotic resistance in rivers, including bacterial communities, human activities, and environmental factors. However, the systematic comparison of the differences in antibiotics resistance and risks between urban rivers (URs) and rural rivers (RRs) in a pharmaceutical industry dominated city is still rare. In this study, Shijiazhuang City (China) was selected as an example to compare the differences in antibiotics resistance and risks between URs and RRs. The results showed higher concentrations of total quinolones (QNs) antibiotics in both water and sediment samples collected from URs than those from RRs. The subtypes and abundances of antibiotic resistance genes (ARGs) in URs were significantly higher than those in RRs, and most emerging ARGs (including OXA-type, GES-type, MCR-type, and tet(X)) were only detected in URs. The ARGs were mainly influenced by QNs in URs and social-economic factors (SEs) in RRs. The composition of the bacterial community was significantly different between URs and RRs. The abundance of antibiotic-resistant pathogenic bacteria (ARPBs) and virulence factors (VFs) were higher in URs than those in RRs. Therein, 371 and 326 pathogen types were detected in URs and RRs, respectively. Most emerging ARGs showed a significantly positive correlation with priority ARPBs. Variance partitioning analysis revealed that SEs were the main driving factors of ARGs (80 %) and microbial communities (92 %) both in URs and RRs. Structural equation models indicated that antibiotics (QNs) and microbial communities were the most direct influence of ARGs in URs and RRs, respectively. The cumulative resistance risk of QNs was high in URs, but relatively low in RRs. Enrofloxacin and flumequine posed the highest risk in water and sediment, respectively. This study could help us to better manage and control the risk of antibiotic resistance in different rivers.
... A: sulfonamide resistance genes; left Y axis: sul1, sul1-Treated, sul2, and sul2-Treated; B: tetracycline resistance genes; left Y axis: tetM and tetM-Treated; C: β-lactam resistance genes; D: macrolide resistance genes; left Y axis: ermB and ermB-Treated; E: aminoglycoside resistance genes; left Y axis: aac (6') -Ib and aac (6') -Ib-Treated; F: quinolone resistance genes; left Y axis: qepA and qepA-Treated; G integrase gene intI1; G removal rate (mean ± SD (5-95% percentile)). of all genes except bla TEM-1 were observed at most time points, whereas the copy numbers of most genes increased markedly at 12:00-20:00 and 20:00-4:00. Plausible explanations of these results included residence time in the septic tank (Buchanan et al., 2018), abrupt anthropogenic input (Lee et al., 2021), and/or desorption from particulate matter (Brown et al., 2019). Previous studies have reported that particulate matter plays a pivotal role in the proliferation and transmission of ARGs during the sewage treatment process (Cui et al., 2020). ...
Article
The temporal variation of antibiotics and ARGs as well as the impact of daily schedule of inpatients on their regular occurrence in hospital wastewater (HWW) were previously obscure. In this study, the wastewater of the inpatient department pre- and posttreatment (hydraulic retention time = 8 h) was collected intraday and intraweek. The absolute concentrations of antibiotics/metabolites and ARGs in HWW were analyzed to investigate the temporal variations of their occurrence levels. Fluoroquinolones were the predominant drugs used in the inpatient department (681.30–881.66 ng/mL in the effluent) and the main contaminant in the outlet of the disinfection pond (538.29–671.47 ng/mL). Diurnal variations peaked at 19:00 for most antibiotics and ARGs, while the maximum of them occurred on weekends. Aminoglycoside resistance genes (AMRGs, 21.6–23000 copies/mL) and β-lactam resistance genes (BLGRs, 1.24–8500 copies/mL) were the dominant ARGs before and after treatment processing, respectively (p < 0.05). The significant removal rates (>50%) of most antibiotics and ARGs, as well as the integrase gene intI1 and 16S rRNA gene, were found to be subjected solely to the chloride disinfection process, suggesting the necessity of the self-contained wastewater treatment process. Meanwhile, the statistically significant correlation among antibiotics, ARGs, intI1, and 16S rRNA (p < 0.05) demonstrated that the risk of selective pressure, horizontal transfer and vertical propagation of ARGs in the effluent of the hospital was warranted. Principal component analysis (PCA) showed that the daily schedule of inpatients and wastewater treatment processes could markedly induce fluctuations in antibiotic and ARG levels in HWW, indicating that they should be considered an impact factor for environmental monitoring. This study demonstrated for the first time the temporal variations in the abundance and dissemination of antibiotics and ARGs in a semiclosed zone and provided new insight into the development of assessments of the associated ecological risk and human health.
... Contrastingly, if functional changes are mainly induced by the release of adapted microorganisms, their spatial extent might be more limited. While for AR the longitudinal effect has recently been shown to decrease rapidly (Lee et al. 2021), such data is missing for biotransformation of MPs. ...
Article
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Biotransformation is the most important process removing manmade chemicals from the environment, yet mechanisms governing this essential ecosystem function are underexplored. To understand these mechanisms, we conducted experiments in flow-through systems, by colonizing stream biofilms under different conditions of mixing river water with treated (and ultrafiltered) wastewater. We performed biotransformation experiments with those biofilms, using a set of 75 micropollutants, and could disentangle potential mechanisms determining the biotransformation potential of stream biofilms. We showed that the increased biotransformation potential downstream of wastewater treatment plants that we observed for specific micropollutants contained in household wastewaters (downstream effect) is caused by microorganisms released with the treated effluent, rather than by the in-stream exposure to those micropollutants. Complementary data from 16S rRNA amplicon-sequencing revealed 146 amplicon sequence variants (ASVs) that followed the observed biotransformation patterns. Our results align with findings for community tolerance, and provide clear experimental evidence that microorganisms released with treated wastewater integrate into downstream biofilms and impact crucial ecosystem functions.
... However, detection of various types of ARGs has been reported in and near wastewater treatment facilities (4)(5)(6)(7)(8), indicating them potential sources for ARGs at the same time. It, unfortunately, seems inevitable as the ARGs may be required and accumulate for the removal of antibiotics in the wastewater from pharmaceutical industries, hospitals, or even municipal sewerage systems, leading to a dilemma in addressing ARG problems. ...
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The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes are key barriers preventing the spread of ARGs, they are often sources of ARGs at the same time, as ARGs may be required and accumulate in the biological treatment units. An upgrading of environmental biotechnology is imperative and urgent. ARGs confer antibiotic resistance based on the DNA sequences rather than the chemistry of DNA molecules. An ARG can be considered degraded if its sequence was disrupted. Therefore, we present here that CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, can be repurposed and tailored for the degradation of ARGs. By deploying an artificial IncP machinery, the designed system, namely VADER, can be successfully delivered via bacterial conjugation. Then, we propose a new sector for ARG degradation to be implemented as a complement to the biological units in the framework of environmental processes. In this endeavor, a prototype conjugation reactor at a 10-mL-scale was devised, and 100% of the target ARG were eliminated in the transconjugated microbes receiving VADER in the reactor. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. Importance Antibiotic resistance has been causing severe health problems and leading to millions of deaths in recent years. Environmental processes, especially the wastewater treatment sector, are important to barrier the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as the source of antibiotic resistance at the same time, as antibiotic resistance with its main cause antibiotic resistance genes (ARGs) may be required and accumulate in the biological treatment units, leading to the dissemination of ARGs. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to environmental biotechnology for tackling the antibiotic resistance dilemma thereof, and we propose a new sector in environmental processes specialized in ARG removal with a reactor inhabiting the CRISPR-Cas system per se. Our study provides a new angle to resolve public health issues via the implementation of synthetic biology at the process level.
... The resistome concept has been recently developed (Bello-López et al., 2019;Lim et al., 2018), including in the case of rivers (Lee et al., 2021;Lekunberri et al., 2018). The resistome is the set of all genes (chromosomic ones responsible for innate resistance and genes acquired by genome changes, which can be inherited in a stable manner from generation to generation or via HGT) that directly or indirectly contribute to AR in the various bacteria (environmental and clinically important pathogens). ...
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River ecosystems are very important parts of the water cycle and an excellent habitat, food, and drinking water source for many organisms, including humans. Antibiotics are emerging contaminants which can enter rivers from various sources. Several antibiotics and their related antibiotic resistance genes (ARGs) have been detected in these ecosystems by various research programs and could constitute a substantial problem. The presence of antibiotics and other resistance cofactors can boost the development of ARGs in the chromosomes or mobile genetic elements of natural bacteria in rivers. The ARGs in environmental bacteria can also be transferred to clinically important pathogens. However, antibiotics and their resistance genes are both not currently monitored by national or international authorities responsible for controlling the quality of water bodies. For example, they are not included in the contaminant list in the European Water Framework Directive or in the US list of Water-Quality Benchmarks for Contaminants. Although ARGs are naturally present in the environment, very few studies have focused on non-impacted rivers to assess the background ARG levels in rivers, which could provide some useful indications for future environmental regulation and legislation. The present study reviews the antibiotics and associated ARGs most commonly measured and detected in rivers, including the primary analysis tools used for their assessment. In addition, other factors that could enhance antibiotic resistance, such as the effects of chemical mixtures, the effects of climate change, and the potential effects of the coronavirus disease 2019 pandemic, are discussed. Environ Toxicol Chem 2022;00:1-27. © 2022 SETAC.
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Climate warming multiplies the threat of antibiotic resistance genes (ARGs) to public health, but whether temperature may predict antibiotic resistomes in water environment remain unknown. Here, by metagenomic sequencing, we investigated the changes of resistome at five different temperature gradients (23, 26, 29, 32, and 35 °C) in polluted water by animal cadaver. Thirty ARG types including 668 subtypes were observed in our samples. Temperature significantly influenced ARG profiles and showed a negative correlation with ARG diversity. The ARG assembly process was dominated by a deterministic process (63.32%–95.08%) but showed a peak pattern with temperature. Notably, temperature may predict approximately 21% of ARGs and 36% of mobile genetic elements (MGEs), while most other ARGs or MGEs were insensitive to temperature. Three types (carbapenem, dicyclomycin, and diaminopyrimidine antibiotic) and 63 subtypes of ARGs that positively correlated with temperature were identified in the polluted water. Notably, we screened 21 subtypes of high-risk ARGs (bacA, mdtA, tetM, etc.) and 22 opportunistic pathogens (Aeromonas, Clostridium, Bacteroides, etc.) and found their positive co-occurrence with temperature, implying these potential biological or genetic pollutants may probably go up under global warming. Our study reveals the predictability of temperature on antibiotic resistance genes, providing a suitable approach to track the fate and spread of ARGs in water environment under climate warming.
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Antibiotic stewardship is hindered by a lack of consideration for complicated environmental fate of antibiotics and their role in resistance development, while the current methodology of eco-toxicological risk assessment has not been fully protective against their potential to select for antibiotic resistance. To address this problem, we established a novel methodologic framework to perform comprehensive environmental risk assessment of antibiotics in terms of resistance development, which was based on selection effect, phenotype resistance level, heteroresistance frequency, as well as prevalence and stability of antibiotic resistance genes. We tracked the contribution of antibiotic load reduction to the mitigation of environmental risk of resistance development by fate and transport modeling. The method was instantiated in a lake-river network-basin complex system, taking the Taihu Basin as a case study. Overall, antibiotic load posed no eco-toxicological risk but an average medium-level environmental risk for resistance development in Taihu Lake. The effect of antibiotic load on resistance risk was both seasonal-dependent and category-dependent, while quinolones posed the greatest environmental risk for resistance development. Mass-flow analysis indicated that temporal-spatial variation in hydrological regime and antibiotic fate together exerted a significant effect on antibiotic load in the system. By apportioning antibiotic load to riverine influx, we identified the hotspots for load reduction and predicted the beneficial response of resistance risk under load-reduction scenarios. Our study proposed a risk-oriented strategy of basin-scaled antibiotic load reduction for environmental risk control of resistance development.
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The large-scale use and release of antibiotics may create selective pressure on antibiotic resistance genes (ARGs), causing potential harm to human health. River ecosystems have long been considered repositories of antibiotics and ARGs. Therefore, the distribution characteristics and seasonal variation in antibiotics and ARGs in the surface water of the main stream and tributaries of the Chishui River were studied. The concentrations of antibiotics in the dry season and rainy season were 54.18–425.74 ng/L and 66.57–256.40 ng/L, respectively, gradually decreasing along the river direction. The results of antibiotics in the dry season and rainy season showed that livestock and poultry breeding were the main sources in the surface water of the Chishui River basin. Risk assessments indicated high risk levels of OFL in both seasons. In addition, analysis of ARGs and microbial community diversity showed that sul1 and sul3 were the main ARGs in the two seasons. The highest abundance of ARGs was 7.70 × 10⁷ copies/L, and intl1 was significantly positively correlated with all resistance genes (p < 0.01), indicating that it can significantly promote the transmission of ARGs. Proteobacteria were the dominant microorganisms in surface water, with a higher average abundance in the dry season (60.64 %) than in the rainy season (39.53 %). Finally, correlation analyses were performed between ARGs and antibiotics, microbial communities and heavy metals. The results showed that there was a significant positive correlation between ARGs and most microorganisms and heavy metals (p < 0.01), indicating that occurrence and transmission in the environment are influenced by various environmental factors and cross-selection. In conclusion, the persistent residue and transmission of ARGs and their transfer to pathogens are a great threat to human health and deserve further study and attention.
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Bacteria associated with atmospheric particulate matter (APMs) can transmit over long distances. A large portion of these bacteria can potentially threaten human health.
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Our decreasing ability to fight bacterial infections is a major health concern. It is arising due to the evolution of antimicrobial resistance (AMR) in response to the mis- and overuse of antibiotics in both human and veterinary medicine. Lakes integrate watershed processes and thus may act as receptors and reservoirs of antibiotic resistance genes (ARGs) introduced into the watershed by human activities. The resistome – the diversity of ARGs – under varying anthropogenic watershed pressures has been previously studied either focused on few select genes or few lakes. Here, we link the resistome of ~350 lakes sampled across Canada to human watershed activity, trophic status, as well as point sources of ARG pollution including wastewater treatment plants and hospitals in the watershed. A high percentage of the resistance genes detected was either unimpacted by human activity or highly prevalent in pristine lakes, highlighting the role of AMR in microbial ecology in aquatic systems, as well as a pool of genes available for potential horizontal gene transfer to pathogenic species. Nonetheless, watershed agricultural and pasture area significantly impacted the resistome. Moreover, the number of hospitals and the population density in a watershed, the volume of wastewater entering the lake, as well as the fraction of manure applied in the watershed as fertilizer significantly impacted ARG diversity. Together, these findings indicate that lake resistomes are regularly stocked with resistance genes evolved in the context of both veterinary and human antibiotics use and represent reservoirs of ARGs that require further monitoring.
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Although triclosan, as a widely used antiseptic chemical, is known to promote the transmission of antibiotic resistance to diverse hosts in pure culture, it is still unclear whether and how triclosan could affect the transmission of broad-host-range plasmids among complex microbial communities. Here, bacterial culturing, fluorescence-based cell sorting, and high-throughput 16S rRNA gene amplicon sequencing were combined to investigate contributions of triclosan on the transfer rate and range of an IncP-type plasmid from a proteobacterial donor to an activated sludge microbiome. Our results demonstrate that triclosan significantly enhances the conjugative transfer of the RP4 plasmid among activated sludge communities at environmentally relevant concentrations. High-throughput 16S rRNA gene sequencing on sorted transconjugants demonstrates that triclosan not only promoted the intergenera transfer but also the intragenera transfer of the RP4 plasmid among activated sludge communities. Moreover, triclosan mediated the transfer of the RP4 plasmid to opportunistic human pathogens, for example, Legionella spp. The mechanism of triclosan-mediated conjugative transfer is primarily associated with excessive oxidative stress, followed by increased membrane permeability and provoked SOS response. Our findings offer insights into the impacts of triclosan on the dissemination of antibiotic resistance in the aquatic environmental microbiome.
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Background The abundance of clinically important antibiotic resistance determinants in wastewater has raised concerns regarding their dissemination into the environment and transmission to animals and humans. However, the high bacterial diversity and complexity of environmental microbiomes makes it challenging to comprehensively detect and quantify clinically relevant resistance determinants. To tackle this outstanding technical bottleneck, classic phenotype-based approaches vastly underestimate the diversity of clinically relevant resistance in environmental reservoirs, while increasingly applied genotyping-based approaches alone usually fail to capture information on the resistance phenotype, viability, and activities of resistant bacteria. Results Here, we developed phenotypic metagenomics as a novel high-throughput approach to couple selective cultivation (phenotyping) and metagenomics (genotyping) to overcome these difficulties of classic phenotype or genotype-based approaches. Using this new method, we were able to track the invasion of wastewater-born multi-resistant opportunistic pathogens into river sediment and epilithic biofilm in 9 Swiss rivers downstream of wastewater treatment plants (WWTPs). The results show that WWTPs release clinically important carbapenem-resistant opportunistic pathogens and promotes the spread of extended-spectrum beta-lactamase resistance genes in the receiving river water. Especially, epilithic biofilms were identified as an unrecognized environmental reservoir of wastewater-associated carbapenem resistant bacteria, which appear to also invade the gut of amphipods, a group of generalist invertebrate in local rivers. We further used metagenome-assembled genomes to characterize phenotypically resistant bacteria and their extended-spectrum beta-lactamase and carbapenemase resistance genes to confirm our finding that wastewater effluent increases resistance levels of extended-spectrum beta-lactamases in the receiving river water. Conclusions This study demonstrates that where culture plating and culture-independent metagenomics fail individually, their complementary combination allows tracking multi-resistant bacteria as they invade river habitats. The proof-of-concept approach that couple resistance phenotyping and genotyping provides a roadmap for sensitive, standardized, and cost-effective surveillance of the clinically relevant aspects of environmental resistomes. The findings call for a thorough life cycle assessment of the selection and transmission of last-resort antibiotic resistance determinants from clinical to environmental reservoirs.
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Background: The impact of human activities on the environmental resistome has been documented in many studies, but there remains the controversial question of whether the increased antibiotic resistance observed in anthropogenically impacted environments is just a result of contamination by resistant fecal microbes or is mediated by indigenous environmental organisms. Here, to determine exactly how anthropogenic influences shape the environmental resistome, we resolved the microbiome, resistome, and mobilome of the planktonic microbial communities along a single river, the Han, which spans a gradient of human activities. Results: The bloom of antibiotic resistance genes (ARGs) was evident in the downstream regions and distinct successional dynamics of the river resistome occurred across the spatial continuum. We identified a number of widespread ARG sequences shared between the river, human gut, and pathogenic bacteria. These human-related ARGs were largely associated with mobile genetic elements rather than particular gut taxa and mainly responsible for anthropogenically driven bloom of the downstream river resistome. Furthermore, both sequence- and phenotype-based analyses revealed environmental relatives of clinically important proteobacteria as major carriers of these ARGs. Conclusions: Our results demonstrate a more nuanced view of the impact of anthropogenic activities on the river resistome: fecal contamination is present and allows the transmission of ARGs to the environmental resistome, but these mobile genes rather than resistant fecal bacteria proliferate in environmental relatives of their original hosts. Video abstract.
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Although Kraken's k-mer-based approach provides a fast taxonomic classification of metagenomic sequence data, its large memory requirements can be limiting for some applications. Kraken 2 improves upon Kraken 1 by reducing memory usage by 85%, allowing greater amounts of reference genomic data to be used, while maintaining high accuracy and increasing speed fivefold. Kraken 2 also introduces a translated search mode, providing increased sensitivity in viral metagenomics analysis.
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Background Infections due to antibiotic-resistant bacteria are threatening modern health care. However, estimating their incidence, complications, and attributable mortality is challenging. We aimed to estimate the burden of infections caused by antibiotic-resistant bacteria of public health concern in countries of the EU and European Economic Area (EEA) in 2015, measured in number of cases, attributable deaths, and disability-adjusted life-years (DALYs). Methods We estimated the incidence of infections with 16 antibiotic resistance–bacterium combinations from European Antimicrobial Resistance Surveillance Network (EARS-Net) 2015 data that was country-corrected for population coverage. We multiplied the number of bloodstream infections (BSIs) by a conversion factor derived from the European Centre for Disease Prevention and Control point prevalence survey of health-care-associated infections in European acute care hospitals in 2011–12 to estimate the number of non-BSIs. We developed disease outcome models for five types of infection on the basis of systematic reviews of the literature. Findings From EARS-Net data collected between Jan 1, 2015, and Dec 31, 2015, we estimated 671 689 (95% uncertainty interval [UI] 583 148–763 966) infections with antibiotic-resistant bacteria, of which 63·5% (426 277 of 671 689) were associated with health care. These infections accounted for an estimated 33 110 (28 480–38 430) attributable deaths and 874 541 (768 837–989 068) DALYs. The burden for the EU and EEA was highest in infants (aged <1 year) and people aged 65 years or older, had increased since 2007, and was highest in Italy and Greece. Interpretation Our results present the health burden of five types of infection with antibiotic-resistant bacteria expressed, for the first time, in DALYs. The estimated burden of infections with antibiotic-resistant bacteria in the EU and EEA is substantial compared with that of other infectious diseases, and has increased since 2007. Our burden estimates provide useful information for public health decision-makers prioritising interventions for infectious diseases.
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Water and sanitation represents a key battlefront in combating the spread of antimicrobial resistance (AMR). Basic water sanitation infrastructure is an essential first step to protecting public health, thereby limiting the spread of pathogens and the need for antibiotics. AMR presents unique human health risks, meriting new risk assessment frameworks specifically adapted to water and sanitation-borne AMR. There are numerous exposure routes to AMR originating from human waste, each of which must be quantified for its relative risk to human health. Wastewater treatment plants (WWTPs) play a vital role in centralized collection and treatment of human sewage, but there are numerous unresolved questions in terms of the microbial ecological processes occurring within and the extent to which they attenuate or amplify AMR. Research is needed to advance understanding of the fate of resistant bacteria and antibiotic resistance genes (ARGs) in various waste management systems, depending on the local constraints and intended re-use applications. WHO and national AMR action plans would benefit from a more holistic 'One Water' understanding. Here we provide a framework for research, policy, practice, and public engagement aimed at limiting the spread of AMR from water and sanitation in both low-, medium- and high-income countries, alike.
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Antibiotics are being used intensively for humans and livestock worldwide and have led to the presence of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. Wastewater treatment plants (WWTPs) have been identified as a point source for ARB&Gs, and water catchments consequently are potential receptors of ARB&Gs. The objective of this study was to investigate the occurrence of antibiotics (macrolides, sulfonamides, tetracyclines), ARGs (ermB, sul1, sul2, tetW), and class 1 integron (targeting the integrase gene), in a Dutch river that receives wastewater treatment plant effluent. Sediment and water samples were collected during one year along the river. The WWTP significantly increased the amounts of antibiotics and ARGs in the river as compared to the upstream samples, of which the antibiotics decreased once they entered the river. ARGs were persistent in the water and sediment from the WWTP effluent discharge point until 20 km downstream. This study provides insight in the prevalence of antibiotics and ARGs in a wastewater effluent-receiving river system in the Netherlands. Even though human antibiotic usage is low in the Netherlands, antibiotics, residues of antibiotics, and ARGs are detected in the river surface water-sediment system, which shows that a river has the potential to act as a reservoir of ARGs.
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Metagenomics emerged as an important field of research not only in microbial ecology but also for human health and disease, and metagenomic studies are performed on increasingly larger scales. While recent taxonomic classification programs achieve high speed by comparing genomic k-mers, they often lack sensitivity for overcoming evolutionary divergence, so that large fractions of the metagenomic reads remain unclassified. Here we present the novel metagenome classifier Kaiju, which finds maximum (in-)exact matches on the protein-level using the Burrows–Wheeler transform. We show in a genome exclusion benchmark that Kaiju classifies reads with higher sensitivity and similar precision compared with current k-mer-based classifiers, especially in genera that are underrepresented in reference databases. We also demonstrate that Kaiju classifies up to 10 times more reads in real metagenomes. Kaiju can process millions of reads per minute and can run on a standard PC. Source code and web server are available at http://kaiju.binf.ku.dk.
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There are concerns that selection pressure from antibiotics in the environment may accelerate the evolution and dissemination of antibiotic-resistant pathogens. Nevertheless, there is currently no regulatory system that takes such risks into account. In part, this is due to limited knowledge of environmental concentrations that might exert selection for resistant bacteria. To experimentally determine minimal selective concentrations in complex microbial ecosystems for all antibiotics would involve considerable effort. In this work, our aim was to estimate upper boundaries for selective concentrations for all common antibiotics, based on the assumption that selective concentrations a priori need to be lower than those completely inhibiting growth. Data on Minimal Inhibitory Concentrations (MICs) were obtained for 111 antibiotics from the public EUCAST database. The 1% lowest observed MICs were identified, and to compensate for limited species coverage, predicted lowest MICs adjusted for the number of tested species were extrapolated through modeling. Predicted No Effect Concentrations (PNECs) for resistance selection were then assessed using an assessment factor of 10 to account for differences between MICs and minimal selective concentrations. The resulting PNECs ranged from 8ng/L to 64μg/L. Furthermore, the link between taxonomic similarity between species and lowest MIC was weak. This work provides estimated upper boundaries for selective concentrations (lowest MICs) and PNECs for resistance selection for all common antibiotics. In most cases, PNECs for selection of resistance were below available PNECs for ecotoxicological effects. The generated PNECs can guide implementation of compound-specific emission limits that take into account risks for resistance promotion.
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Antibiotic resistance is a threat to human and animal health worldwide, and key measures are required to reduce the risks posed by antibiotic resistance genes that occur in the environment. These measures include the identification of critical points of control, the development of reliable surveillance and risk assessment procedures, and the implementation of technological solutions that can prevent environmental contamination with antibiotic resistant bacteria and genes. In this Opinion article, we discuss the main knowledge gaps, the future research needs and the policy and management options that should be prioritized to tackle antibiotic resistance in the environment.
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Antibiotic-resistance genes (ARGs) are currently discussed as emerging environmental contaminants. Hospital and municipal sewage are important sources of ARGs for the receiving freshwater bodies. We investigated the spatial distribution of different ARGs (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) in freshwater lake sediments in the vicinity of a point source of treated wastewater. ARG contamination of Vidy Bay, Lake Geneva, Switzerland was quantified using real-time PCR and compared with total mercury (THg), a frequently particle-bound inorganic contaminant with known natural background levels. Two-dimensional mapping of the investigated contaminants in lake sediments with geostatistical tools revealed total and relative abundance of ARGs in close proximity of the sewage discharge point were up to 200-fold above levels measured at a remote reference site (center of the lake) and decreased exponentially with distance. Similar trends were observed in the spatial distribution of different ARGs, whereas distributions of ARGs and THg were only moderately correlated, indicating differences in the transport and fate of these pollutants or additional sources of ARG contamination. The spatial pattern of ARG contamination and supporting data suggest that deposition of particle-associated wastewater bacteria rather than co-selection by, for example, heavy metals was the main cause of sediment ARG contamination.
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Reference genomes are required to understand the diverse roles of microorganisms in ecology, evolution, human and animal health, but most species remain uncultured. Here we present a sequence composition-independent approach to recover high-quality microbial genomes from deeply sequenced metagenomes. Multiple metagenomes of the same community, which differ in relative population abundances, were used to assemble 31 bacterial genomes, including rare (<1% relative abundance) species, from an activated sludge bioreactor. Twelve genomes were assembled into complete or near-complete chromosomes. Four belong to the candidate bacterial phylum TM7 and represent the most complete genomes for this phylum to date (relative abundances, 0.06-1.58%). Reanalysis of published metagenomes reveals that differential coverage binning facilitates recovery of more complete and higher fidelity genome bins than other currently used methods, which are primarily based on sequence composition. This approach will be an important addition to the standard metagenome toolbox and greatly improve access to genomes of uncultured microorganisms.
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One of the currently most relevant resistance mechanisms in Enterobacteriaceae is the production of enzymes that lead to higher generation cephalosporin and even carbapenem resistance, mainly extended-spectrum beta-lactamases (ESBL) and carbapenemases. A worrisome aspect is the spread of ESBL- and carbapenemase-producers into the environment. The aim of the present study was to assess the occurrence of, and to further characterize ESBL- and carbapenemase producing Enterobacteriaceae in rivers and lakes in Switzerland. ESBL-producing Enterobacteriaceae were detected in 21 (36.2%) of the 58 sampled water bodies. One river sample tested positive for a carbapenemase-producing Klebsiella penumoniae ssp. pneumoniae strain. Seventy-four individual strains expressing an ESBL phenotype were isolated. Species identification revealed sixty representatives of Escherichia coli, seven Klebsiella penumoniae ssp. pneumoniae, five Raoultella planticola, one Enterobacter cloacae and one E. amnigenus. Three strains were identified as SHV-12 ESBL producers, and 71 strains carried genes encoding for CTX-M ESBLs. Eight isolates among the latter expressed CTX-M-1, three produced CTX-M-3, 46 produced CTX-M-15, three produced CTX-M-55, one produced CTX-M-79, six produced CTX-M-14 and four produced CTX-M-27. Three of the four CTX-M-27 producers belonged to the multi-resistant pandemic sequence type E. coli B2:ST131 that is strongly associated with potentially severe infections in humans and animals.
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The use of antibiotic agents as growth promoters was banned in animal husbandry to prevent the selection and spread of antibiotic resistance. However, in addition to antibiotic agents, heavy metals used in animal farming and aquaculture might promote the spread of antibiotic resistance via co-selection. To investigate which heavy metals are likely to co-select for antibiotic resistance in soil and water, the available data on heavy metal pollution, heavy metal toxicity, heavy metal tolerance, and co-selection mechanisms was reviewed. Additionally, the risk of metal driven co-selection of antibiotic resistance in the environment was assessed based on heavy metal concentrations that potentially induce this co-selection process. Analyses of the data indicate that agricultural and aquacultural practices represent major sources of soil and water contamination with moderately to highly toxic metals such as mercury (Hg), cadmium (Cd), copper (Cu), and zinc (Zn). If those metals reach the environment and accumulate to critical concentrations they can trigger co-selection of antibiotic resistance. Furthermore, co-selection mechanisms for these heavy metals and clinically as well as veterinary relevant antibiotics have been described. Therefore, studies investigating co-selection in environments impacted by agriculture and aquaculture should focus on Hg, Cd, Cu, and Zn as selecting heavy metals. Nevertheless, the respective environmental background has to be taken into account.
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At present, very little is known about the fate and persistence of multiresistant bacteria (MRB) and their resistance genes in natural aquatic environments. Treated, but partly also untreated sewage of the city of Lausanne, Switzerland is discharged into Vidy Bay (Lake Geneva) resulting in high levels of contamination in this part of the lake. In the present work we have studied the prevalence of MRB and resistance genes in the wastewater stream of Lausanne. Samples from hospital and municipal raw sewage, treated effluent from Lausanne's wastewater treatment plant (WTP) as well as lake water and sediment samples obtained close to the WTP outlet pipe and a remote site close to a drinking water pump were evaluated for the prevalence of MRB. Selected isolates were identified (16S rRNA gene fragment sequencing) and characterized with regards to further resistances, resistance genes, and plasmids. Mostly, studies investigating this issue have relied on cultivation-based approaches. However, the limitations of these tools are well known, in particular for environmental microbial communities, and cultivation-independent molecular tools should be applied in parallel in order to take non-culturable organisms into account. Here we directly quantified the sulfonamide resistance genes sul1 and sul2 from environmental DNA extracts using TaqMan real-time quantitative PCR. Hospital sewage contained the highest load of MRB and antibiotic resistance genes (ARGs). Wastewater treatment reduced the total bacterial load up to 78% but evidence for selection of extremely multiresistant strains and accumulation of resistance genes was observed. Our data clearly indicated pollution of sediments with ARGs in the vicinity of the WTP outlet. The potential of lakes as reservoirs of MRB and potential risks are discussed.
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As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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The use of antibiotics may accelerate the development of antibiotic resistance genes (ARGs) and bacteria which shade health risks to humans and animals. The emerging of ARGs in the water environment is becoming an increasing worldwide concern. Hundreds of various ARGs encoding resistance to a broad range of antibiotics have been found in microorganisms distributed not only in hospital wastewaters and animal production wastewaters, but also in sewage, wastewater treatment plants, surface water, groundwater, and even in drinking water. This review summarizes recently published information on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs, including specific and multiplex PCR (polymerase chain reaction), real-time PCR, DNA sequencing, and hybridization based techniques.
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Class 1 integrons are central players in the worldwide problem of antibiotic resistance, because they can capture and express diverse resistance genes. In addition, they are often embedded in promiscuous plasmids and transposons, facilitating their lateral transfer into a wide range of pathogens. Understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring how lateral gene transfer can seriously impact on, and be impacted by, human activities. We now show that class 1 integrons can be found on the chromosomes of nonpathogenic soil and freshwater Betaproteobacteria. Here they exhibit structural and sequence diversity, an absence of antibiotic resistance genes, and a phylogenetic signature of lateral transfer. Some examples are almost identical to the core of the class 1 integrons now found in pathogens, leading us to conclude that environmental Betaproteobacteria were the original source of these genetic elements. Because these elements appear to be readily mobilized, their lateral transfer into human commensals and pathogens was inevitable, especially given that Betaproteobacteria carrying class 1 integrons are common in natural environments that intersect with the human food chain. The strong selection pressure imposed by the human use of antimicrobial compounds then ensured their fixation and global spread into new species.
Article
Wastewater treatment plant (WWTP) effluents release not only chemical constituents in watersheds, but also contain microorganisms. Thus, an understanding of what microorganisms are released and how they change microbial communities within natural streams is needed. To characterize the community shifts in streams receiving WWTP effluent, we sampled water-column microorganisms from upstream, downstream, and the effluent of WWTPs located on 23 headwater streams in which no other effluent was released upstream. We characterized the bacterial community by sequencing the V3-V4 region of the 16S rRNA gene. We hypothesized that the downstream community profile would be a hydraulic mixture between the two sources (i.e., upstream and effluent). In ordination analyses, the downstream bacterial community profile was a mixture between the upstream and effluent. For 14 of the sites, the main contribution (>50%) to the downstream community originated from bacteria in the WWTP effluent and significant shifts in relative abundance of specific sequence variants were detected. These shifts in sequence variants may serve as general bioindicators of wastewater-effluent influenced streams, with a human-gut related Ruminococcus genus displaying the highest shift (30-fold higher abundances downstream). However, not all taxa composition changes were predicted based on hydraulic mixing alone. Specifically, the decrease of Cyanobacteria/Chloroplast reads was not adequately described by hydraulic mixing. The potential alteration of stream microbial communities via a high inflow of human-gut related bacteria and a decrease in autotrophic functional groups resulting from WWTP effluent creates the potential for general shifts in stream ecosystem function.
Article
Wastewater treatment plants (WWTPs) are implicated as hotspots for the dissemination of antibacterial resistance into the environment. However, the in situ processes governing removal, persistence, and evolution of resistance genes during wastewater treatment remain poorly understood. Here, we used quantitative metagenomic and metatranscriptomic approaches to achieve a broad-spectrum view of the flow and expression of genes related to antibacterial resistance to over 20 classes of antibiotics, 65 biocides, and 22 metals. All compartments of 12 WWTPs share persistent resistance genes with detectable transcriptional activities that were comparatively higher in the secondary effluent, where mobility genes also show higher relative abundance and expression ratios. The richness and abundance of resistance genes vary greatly across metagenomes from different treatment compartments, and their relative and absolute abundances correlate with bacterial community composition and biomass concentration. No strong drivers of resistome composition could be identified among the chemical stressors analyzed, although the sub-inhibitory concentration (hundreds of ng/L) of macrolide antibiotics in wastewater correlates with macrolide and vancomycin resistance genes. Contig-based analysis shows considerable co-localization between resistance and mobility genes and implies a history of substantial horizontal resistance transfer involving human bacterial pathogens. Based on these findings, we propose future inclusion of mobility incidence (M%) and host pathogenicity of antibiotic resistance genes in their quantitative health risk ranking models with an ultimate goal to assess the biological significance of wastewater resistomes with regard to disease control in humans or domestic livestock.
Article
Motivation: Much global attention has been paid to antibiotic resistance in monitoring its emergence, accumulation and dissemination. For rapid characterization and quantification of antibiotic resistance genes (ARGs) in metagenomic datasets, an online analysis pipeline, ARGs-OAP has been developed consisting of a database termed Structured Antibiotic Resistance Genes (the SARG) with a hierarchical structure (ARGs type-subtype-reference sequence). Results: The new release of the database, termed SARG version 2.0, contains sequences not only from CARD and ARDB databases, but also carefully selected and curated sequences from the latest protein collection of the NCBI-NR database, to keep up to date with the increasing number of ARG deposited sequences. SARG v2.0 has tripled the sequences of the first version and demonstrated improved coverage of ARGs detection in metagenomes from various environmental samples. In addition to annotation of high-throughput raw reads using a similarity search strategy, ARGs-OAP v2.0 now provides model-based identification of assembled sequences using SARGfam, a high-quality profile Hidden Markov Model (HMM), containing profiles of ARG subtypes. Additionally, ARGs-OAP v2.0 improves cell number quantification by using the average coverage of essential single copy marker genes, as an option in addition to the previous method based on the 16S rRNA gene. Availability: ARGs-OAP can be accessed through http://smile.hku.hk/SARGs. The database could be downloaded from the same site. Source codes for this study can be downloaded from https://github.com/xiaole99/ARGs-OAP-v2.0. Contact: zhangt@hku.hk; tiedjej@msu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.
Chapter
Human-induced environmental changes are causing major shifts in ecosystems around the globe. To support environmental management, scientific research has to infer both general trends and context dependency in these shifts at global and local scales. Combining replicated . real-world experiments, which take advantage of implemented mitigation measures or other forms of human impact, with . research-led experimental manipulations can provide powerful scientific tools for inferring causal drivers of ecological change and the generality of their effects. Additionally, combining these two approaches can facilitate communication with stakeholders involved in implementing management strategies. We demonstrate such an integrative approach using the case study . EcoImpact, which aims at empirically unravelling the impacts of wastewater-born micropollutants on aquatic ecosystems.
Article
We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.
Article
Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) in the aquatic environment have become an emerging contaminant issue, which has implications for human and ecological health. This review begins with an introduction to the occurrence of ARB and ARG in different environmental systems such as natural environments and drinking water resources. For example, ARG or ARB with resistance to ciprofloxacin, sulfamethoxazole, trimethoprim, quinolone, vancomycin, or tetracycline (e.g., tet(A), tet(B), tet(C), tet(G), tet(O), tet(M), tet(W), sul I, and sul II) have been detected in the environment. The development of resistance may be intrinsic, may be acquired through spontaneous mutations (de novo), or may occur due to horizontal gene transfer from donor bacteria, phages, or free DNA to recipient bacteria. An overview is also provided of the current knowledge regarding inactivation of ARB and ARG, and the mechanism of the effects of different disinfection processes in water and wastewater (chlorination, UV irradiation, Fenton reaction, ozonation, and photocatalytic oxidation). The effects of constructed wetlands and nanotechnology on ARB and ARG are also summarized.
Article
Urban wastewater treatment plants (UWTPs) are among the main sources of antibiotics' release into the environment. The occurrence of antibiotics may promote the selection of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), which shade health risks to humans and animals. In this paper the fate of ARB and ARGs in UWTPs, focusing on different processes/technologies (i.e., biological processes, advanced treatment technologies and disinfection), was critically reviewed. The mechanisms by which biological processes influence the development/selection of ARB and ARGs transfer are still poorly understood. Advanced treatment technologies and disinfection process are regarded as a major tool to control the spread of ARB into the environment. In spite of intense efforts made over the last years to bring solutions to control antibiotic resistance spread in the environment, there are still important gaps to fill in. In particular, it is important to: (i) improve risk assessment studies in order to allow accurate estimates about the maximal abundance of ARB in UWTPs effluents that would not pose risks for human and environmental health; (ii) understand the factors and mechanisms that drive antibiotic resistance maintenance and selection in wastewater habitats. The final objective is to implement wastewater treatment technologies capable of assuring the production of UWTPs effluents with an acceptable level of ARB.
Article
Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a consequence of weak, non-lethal selective pressures - caused by low levels of antibiotics - that operates on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant appearance and promotes enrichment of highly fit mutants and stable mutators.
Article
A novel approach utilizing antibiotic-resistance-gene (ARG) molecular signatures was applied to track the sources of ARGs at sites along the Cache la Poudre (Poudre) and South Platte Rivers in Colorado. Two lines of evidence were employed: (1) detection frequencies of 2 sulfonamide and 11 tetracycline ARGs and (2) tet(W) phylotype and phylogenetic analysis. A GIS database indicating the locations of wastewater treatment plants (WWTPs) and animal feeding operations (AFOs) in the watershed was also constructed to assess congruence of the surrounding landscape with the putative sources identified by ARG molecular signatures. Discriminant analysis was performed on detection frequencies of tetARG groups that were previously identified to be associated with either WWTPs or AFOs. All but one (South Platte River-3, just downstream from the confluence with the Poudre River) of the eight sites were classified as primarily WWTP-influenced based on discriminant analysis of ARG detection frequencies. tet(W) phylotype analysis also aligned South Platte River-3 with putative AFO sources, while phylogenetic analysis indicated that it was not significantly different from the AFOs or WWTPs investigated. South Platte River-3 is situated in an intense agricultural area, but the upstream portion of the South Platte River receives substantial loading from metropolitan Denver. By contrast, tet(W) phylotype and phylogenetics of site Poudre River-4, located 4 km downstream of a WWTP, was also characterized and found to be significantly different from the AFO lagoons (p < 0.05), as expected. In general, a good correspondence was found between classification of the impacted river sites and the surrounding landscape. While the overall approach could be extended to other watersheds, the general findings indicate that transport of ARGs from specific sources is likely the dominant mechanism for ARG proliferation in this riverine environment relative to selection of ARGs among native bacteria by antibiotics and other pollutants.
Article
Biocides and pesticides are designed to control the occurrence of unwanted organisms. From their point of application, these substances can be mobilized and transported to surface waters posing a threat to the aquatic environment. Historically, agricultural pesticides have received substantially more attention than biocidal compounds from urban use, despite being used in similar quantities. This study aims at improving our understanding of the influence of mixed urban and agricultural land use on the overall concentration dynamics of biocides and pesticides during rain events throughout the year. A comprehensive field study was conducted in a catchment within the Swiss plateau (25 km(2)). Four surface water sampling sites represented varying combinations of urban and agricultural sources. Additionally, the urban drainage system was studied by sampling the only wastewater treatment plant (WWTP) in the catchment, a combined sewer overflow (CSO), and a storm sewer (SS). High temporal resolution sampling was carried out during rain events from March to November 2007. The results, based on more than 600 samples analyzed for 23 substances, revealed distinct and complex concentration patterns for different compounds and sources. Five types of concentration patterns can be distinguished: a) compounds that showed elevated background concentrations throughout the year (e.g. diazinon >50 ng L(-1)), indicating a constant household source; b) compounds that showed elevated concentrations driven by rain events throughout the year (e.g. diuron 100-300 ng L(-1)), indicating a constant urban outdoor source such as facades; c) compounds with seasonal peak concentrations driven by rain events from urban and agricultural areas (e.g. mecoprop 1600 ng L(-1) and atrazine 2500 ng L(-1) respectively); d) compounds that showed unpredictably sharp peaks (e.g. atrazine 10,000 ng L(-1), diazinon 2500 ng L(-1)), which were most probably due to improper handling or even disposal of products; and finally, e) compounds that were used in high amounts but were not detected in surface waters (e.g. isothiazolinones). It can be safely concluded that in catchments of mixed land use, the contributions of biocide and pesticide inputs into surface waters from urban areas are at least as important as those from agricultural areas.
Article
The genus Pseudomonas was described in 1894 and is one of the most diverse and ubiquitous bacterial genera whose species have been isolated worldwide in all kinds of environments, from the Antarctica to the Tropics, present in sediments, clinical samples, plant, fungi and diseased animal specimens, water, soil, plant rhizosphere, sea, deserts, etc. The taxonomy of the genus has been controversial for years since a lot of bacterial taxa initially included in genus Pseudomonas have been reclassified in other genera or species from a different class of Proteobacteria over the years, as techniques for characterization and classification of microorganisms improved, aiming to set a phylogenetic classification of the species. In this review, the historical evolution of the taxonomy of Pseudomonas is described, and the currently valid criteria and future challenges for taxonomy of the genus and techniques used to achieve the necessary characterization for classifying the species are discussed. Finally, all the validly published Pseudomonas species at present are listed with an overview of their diversity and ecology.
Article
The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.
Article
The heavy use of veterinary antibiotics in modern animal production causes concern about risks of spreading antibiotic resistance after manure applications to agricultural fields. We report on a field study aiming at elucidating the fate of sulfonamide (SA) antibiotics in grassland soils and their transport to surface water. Two controlled manure applications were carried out under different weather conditions. After both applications, the SA concentrations in pore water and the total soil content declined rapidly. This stage of fast decline was followed by a second one during which the SA were rather persistent. More than 15% of the SAs applied were still present in the soil 3 months after application, always exceeding 100 microg/kg topsoil. The apparent SA sorption increased strongly with time. Accordingly, the risk for SA losses to water bodies decreased within 2 weeks to very low values. In contrast to SA concentrations in the soil, losses to the brook were strongly influenced by the weather conditions after the two manure applications. The overall losses were 15 times larger (about 0.5% of applied SA) during the wet conditions of May 2003 compared to the dry conditions following the first application (March 2003).
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