This study quantified antibiotic and antibiotic resistance gene (ARG) concentrations in hospital and communal wastewaters as well as the influents and effluents of the receiving urban wastewater ...treatment plants (UWWTP) in two Dutch cities. In only one city, hospital wastewater was treated on-site using advanced technologies, including membrane bioreactor treatment (MBR), ozonation, granulated activated carbon (GAC) and UV-treatment.
On-site hospital wastewater (HWW) treatment reduced gene presence of hospital-related antibiotic resistance genes and antibiotic concentrations in the receiving urban wastewater treatment plant. These findings support the need for on-site treatment of high-risk point sources of antibiotic resistance genes.
13 antibiotic resistance genes, Integrase Class 1 and 16S rRNA concentrations were quantified using multiplex quantitative real-time PCR (qPCR) assays and the presence and/or concentration of 711 antibiotics were analyzed.
Hospital wastewater contained approximately 25% more antibiotics and gene concentrations between 0.4 log to 1.8-fold higher than communal wastewater (CWW). blaKPC and vanA could be identified as hospital-related genes and were reduced to under the limit of detection (LOD) during on-site treatment. Advanced on-site treatment removed between 0.5 and 3.6-fold more genes than conventional biological urban wastewater treatment (activated sludge). Advanced on-site treatment was able to eliminate 12 out of 19 detected antibiotics, while urban waste water treatment eliminated up to 1 (out of 21 detected). Different advanced treatment technologies were able to target different pollutants to varying extents, making sequential alignment more effective. MBR treatment was most efficient in antibiotic resistance gene reduction and ozonation in antibiotic reduction.
blaKPC could only be detected in the influent of the urban wastewater treatment plant receiving untreated hospital wastewater. Similarly, vanA was only consistently detected in this treatment plant. These results indicate a positive effect of on-site treatment of hospital wastewater on the communal sewage system.
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Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, ...Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobiotic metabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.
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•280 out of 2248 target substances were detected.•Detected chemicals were evaluated based on their ecotoxicological properties.•10 in vitro bioassays were applied for assessing the adverse effects in wastewater.•Effect-based risk assessment of WWTP effluents was applied.•13 antibiotic resistant genes were determined in the Danube River Basin.
Social concern has raised during the last years due to the development of antibiotic resistance hotspots in different environmental compartments, including the edible parts of crops. To assess the ...influence of the water quality used for watering, we collected samples from soil, roots, leaves and beans from the legume plant Vicia faba (broad beans) in three agricultural peri-urban plots (Barcelona, NE Spain), irrigated with either groundwater, river water, or reclaimed water. Antibiotic resistance genes (ARGs) sul1, tetM, qnrS1, blaCTX-M-32,blaOXA-58, mecA, and blaTEM were quantified by real-time PCR, along with 16S rDNA and intl1 sequences, as proxies for bacterial abundance and integron prevalence, respectively. Microbiome composition of all samples were analyzed by high-throughput DNA sequencing. Results show a gradient of bacterial species diversity and of ARG prevalence from highly diverse soil samples to microbially-poor beans and leaves, in which Rhizobiales essentially displaced all other groups, and that presented very small loads of ARGs and integron sequences. The data suggest that the microbiome and the associated resistome were likely influenced by agricultural practices and water quality, and that future irrigation water legal standards should consider the specific Physiology of the different crop plants.
•ARGs presented different abundance profiles according to the sampling fields.•Rhizobiales dominate the microbiome of all legume plant parts.•intl1 and ARGs abundances inversely correlate with Rhizobiales' predominance.•Rhizobiales roots’ colonization influences the microbiome of surrounding soil.
The aim of this study was to capture and explain changes in antibiotic resistance gene (ARG) presence and concentration internationally across the Rhine river. Intl1 concentrations and national ...antibiotic usage were investigated as proxies to predict anthropogenic ARG pollution. Newly-developed multiplex qPCR assays were employed to investigate ARG profiles across 8 locations (L1-L8) in three countries (Switzerland, Germany, the Netherlands) and to detect potential regional causes for variation. Two of these locations were further monitored, over the duration of one month. A total of 13 ARGs, Intl1 and 16S rRNA were quantified.
ARG presence and concentrations initially increased from L1(Diepoldsau) to L3(Darmstadt). A continuous increase could not be observed at subsequent locations, with the large river volume likely being a major contributing factor for stability. ARG presence and concentrations fluctuated widely across different locations. L2(Basel) and L3 were the two most polluted locations, coinciding with these locations being well-developed pharmaceutical production locations.
We draw attention to the characteristic, clearly distinct ARG profiles, with gene presence being consistent and gene concentrations varying significantly less over time than across different locations. Five genes were Rhine-typical (ermB, ermF, Intl1, sul1 and tetM). Intl1 and sul1 were the genes with highest and second-highest concentration, respectively. Aph(III)a and blaOXA were permanently introduced downstream of L1, indicating no source of these genes prior to L1.
We highlight that correlations between Intl1 and ARG concentrations (R2 = 0.72) were driven by correlations to sul1 and disappeared when excluding sul1 from the analysis (R2 = 0.05). Intl1 therefore seems to be a good proxy for sul1 concentrations but not necessarily for overall (anthropogenic) ARG pollution. Aminoglycoside usage per country correlated with concentrations of aph(III)a and several unrelated antibiotic resistance genes (blaOXA,ermB, ermF and tetM). This correlation can be explained by co-resistance caused by mobile genetic elements (MGEs), such as Tn1545.
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•Five multiplex qPCR assays were developed for monitoring ARGs in environmental samples.•Rhine river locations have distinct, characteristic antibiotic resistance genes (ARG) profiles.•ARG concentration do not continuously increase at more downstream locations.•Intl1 correlates with overall ARG concentrations only due to correlations with sul1.•ARG concentrations do not correlate with regional antibiotic use.
Despite the social concern about the generalization of antibiotic resistance hotspots worldwide, very little is known about the contribution of different potential sources to the global risk. Here we ...present a quantitative analysis of the distribution of Antibiotic Resistance Genes (ARGs) in soil, rhizospheric soil, roots, leaves and beans in tomato, lettuce and broad beans crops (165 samples in total), grown in nine commercial plots distributed in four geographical zones in the vicinity of Barcelona (North East Spain). We also analyzed five soil samples from a nearby forest, with no record of agricultural activities. DNA samples were analyzed for their content in the ARGs sul1, tetM, qnrS1, blaCTX-M-32, blaOXA-58, mecA, and blaTEM, plus the integron intI1, using qPCR methods. In addition, soil microbiomes from the different plots were analyzed by amplicon-targeted 16S rRNA gene sequencing. Our data show a decreasing gradient of ARG loads from soil to fruits and beans, the latter showing only from 0.1 to 0.01% of the abundance values in soil. The type of crop was the main determinant for both ARG distribution and microbiome composition among the different plots, with minor contributions of geographic location and irrigation water source. We propose that soil amendment and/or fertilization, more than irrigation water, are the main drivers of ARG loads on the edible parts of the crop, and that they should therefore be specifically controlled.
•Antibiotic Resistance Gene (ARG) distribution in commercial plants and plots.•A gradient of ARG loads from soil (most) to roots, leaves, fruits, and beans (least).•Crop type determinant for ARG distribution and microbiome composition.•Minimal effect of irrigation water quality on ARG abundance in soils and plants.
To ensure delivery of microbiologically safe drinking water, the physical integrity of the distribution system is an important control measure. During repair works or an incident the drinking water ...pipe is open and microbiologically contaminated water or soil may enter. Before taking the pipe back into service it must be cleaned. The efficacy of flushing and shock chlorination was tested using a model pipe-loop system with a natural or cultured biofilm to which a microbial contamination (Escherichia coli, Clostridium perfringens spores and phiX174) was added. On average, flushing removed 1.5–2.7 log microorganisms from the water, but not the biofilm. In addition, sand added to the system was not completely removed. Flushing velocity (0.3 or 1.5 m/s) did not affect the efficacy. Shock chlorination (10 mg/L, 1–24 h) was very effective against E. coli and phiX174, but C. perfringens spores were partly resistant. Chlorination was slightly more effective in pipes with a natural compared to a cultured biofilm. Flushing alone is thus not sufficient after high risk repair works or incidents, and shock chlorination should be considered to remove microorganisms to ensure microbiologically safe drinking water. Prevention via hygienic working procedures, localizing and isolating the contamination source and issuing boil water advisories remain important, especially during confirmed contamination events.
Soil passage of (pretreated) surface water to remove pathogenic microorganisms is a highly efficient process under oxic conditions, reducing microorganism concentrations about 8 log10 within tens of ...meters. However, under anoxic conditions, it has been shown that removal of microorganisms can be limited very much. Setback distances for adequate protection of natural groundwater may, therefore, be too short if anoxic conditions apply. Because removal of microorganisms under suboxic conditions is unknown, this research investigated removal of bacteriophage MS2 and PRD1 by soil passage under suboxic conditions at field scale. At the field location (dune area), one injection well and six monitoring wells were installed at different depths along three suboxic flow lines, where oxygen concentrations ranged from 0.4 to 1.7 mg/l and nitrate concentrations ranged from 13 to 16 mg/L. PRD1 and MS2 were injected directly at the corresponding depths and their removal in each flow line was determined. The highest bacteriophage removal was observed in the top layer, with about 9 log removal of MS2, and 7 log removal of PRD1 after 16 meters of aquifer transport. Less removal was observed at 12 m below surface, probably due to a higher groundwater velocity in this coarser grained layer. MS2 was removed more effectively than PRD1 under all conditions. Due to short travel times, inactivation of the phages was limited and the reported log removal was mainly associated with attachment of phages to the aquifer matrix. This study shows that attachment of MS2 and PRD1 is similar for oxic and suboxic sandy aquifers, and, therefore, setback distances used for sandy aquifers under oxic and suboxic conditions provide a similar level of safety. Sticking efficiency and the attachment rate coefficient, as measures for virus attachment, were evaluated as a function of the physico-chemical conditions.
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•Virus removal in a suboxic aquifer is equally effective as in an oxic aquifer.•16 m of suboxic aquifer transport removed MS2 by 9 log10 and PRD1 by 7 log10.•Under suboxic conditions a travel time of 60 days is adequate for protection of groundwater.
Bacterial spores are the ultimate (stress) ‘survival capsules’. They allow strains from the
Bacillus and
Clostridium species to survive harsh environmental conditions. In addition to the decision to ...enter sporulation the decision to do the reverse (germinate) is also a decisive event after which there is no return. Generally it is observed that the behaviour of spores towards the environment is not homogeneous. In fact in many cases it is even quite heterogeneous, certainly upon subjecting the spores to a thermal stress treatment. Genome information coupled to high resolution single-cell analysis techniques allow us currently to analyse signalling events of individual cells. In the area of food preservation the next challenge is to couple the newly acquired mechanistic data to the physiologically observed heterogeneity in spore behaviour.
The current paper will introduce the background of physiological heterogeneity while discussing the molecular processes that likely contribute to the observed heterogeneity in outgrowth. The discussion is set in the framework of contemporary and future needs for single-cell data integration in order to enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores.
Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of Bacillus cereus ATCC 14579. A survey including 20 different amino ...acids showed that L-alanine, L-cysteine, L-threonine, and L-glutamine are capable of initiating the germination of endospores of B. cereus ATCC 14579. In addition, the purine ribonucleosides inosine and adenosine can trigger germination of the spores. Advanced annotation of the B. cereus ATCC 14579 genome revealed the presence of seven putative germination (ger) operons, termed gerG, gerI, gerK, gerL, gerQ, gerR, and gerS. To determine the role of the encoded putative receptors in nutrient-induced germination, disruption mutants were constructed by the insertion of pMUTIN4 into each of the seven operons. Four of the seven mutants were affected in the germination response to amino acids or purine ribonucleosides, whereas no phenotype could be attributed to the mutants with disrupted gerK, gerL, and gerS loci. The strain with a disrupted gerR operon was severely hampered in the ability to germinate: germination occurred in response to L-glutamine but not in the presence of any of the other amino acids tested. The gerG mutant showed significantly reduced L-glutamine-induced germination, which points to a role of this receptor in the L-glutamine germination signaling pathway. gerR, gerI, and gerQ mutants showed reduced germination rates in the presence of inosine, suggesting a role for these operons in ribonucleoside signaling. Efficient germination by the combination of L-glutamine and inosine was shown to involve the gerG and gerI operons, since the germination of mutants lacking either one of these receptors was significantly reduced. Germination triggered by the combination of L-phenylalanine and inosine was lost in the gerI mutant, indicating that both molecules are effective at the GerI receptor.
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