Norbert Kreuzinger

Tackling antibiotic resistance: the environmental framework

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.

Micropollutant removal during biological wastewater treatment and a subsequent ozonation step.

The design criteria for wastewater treatment plants (WWTP) and the sludge retention time, respectively, have a significant impact on micropollutant removal. The upgrade of an Austrian municipal WWTP to nitrogen removal (best available technology, BAT) resulted in increased elimination of most of the analyzed micropollutants. Substances, such as bisphenol-A, 17alpha-ethinylestradiol and the antibiotics erythromycin and roxithromycin were only removed after the upgrade of the WWTP. Nevertheless, the BAT was not sufficient to completely eliminate these compounds. Thus, a pilot scale ozonation plant was installed for additional treatment of the effluent. The application of 0.6 g O(3) g DOC(-1) increased the removal of most of the micropollutants, especially for compounds that were not degraded in the previous biological process, as for example carbamazepine and diclofenac. These results indicated that the ozonation of WWTP effluent is a promising technology to further decrease emissions of micropollutants from the treatment process.

Simultaneous detection and differentiation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene.

ABSTRACT A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the β-d-glucuronidase gene (uidA) for specific detection and differentiation of Escherichia colipopulations according to their uidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. colifreshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.

Impact of ozonation on the genotoxic activity of tertiary treated municipal wastewater

Ozonation is an emerging technology for the removal of micropollutants from treated wastewater. Aim of the present study was to investigate the impact of ozone treatment on genotoxic and acute toxic effects of tertiary treated municipal wastewater. It is known that DNA-damaging chemicals cause adverse effects in the environment and that exposure to humans leads to cancer and other diseases. Toxicity was tested in organisms from three trophic levels namely in bacteria (Salmonella/microsome assays) which enable the detection of gene mutations, in a plant bioassay (micronucleus assay with root tip cells of Allium cepa) which reflects clastogenic and aneugenic effects and in single cell gel electrophoresis (SCGE) tests with mammalian cells which detect DNA migration caused by single-, double strand breaks and alkali labile sites. In the bacterial tests negative results were obtained with untreated samples but after concentration with C(18) cartridges a positive result was found in strains TA1537 and TA98 which are sensitive to frameshift mutagens while no mutations were induced in other tester strains (TA100, TA102 and YG1024). Ozone treatment led to a decrease of the mutagenic activity of the samples. In the SCGE experiments, DNA migration was detected with the unconcentrated effluent of the treatment plant and ozonation led to a substantial decrease of this effect. In the plant bioassays, negative results were obtained with the effluent and ozone treatment did not cause an alteration of the micronucleus frequencies. Also acute toxic effects were monitored in the different indicator organisms under all experimental conditions. The bacteriocidal/bacteriostatic effects which were seen with the concentrated samples were reduced by ozonation. In the experiments with the eukaryotic (plant and animal) cells no acute toxicity was seen with the effluents and ozonation had no impact on their viability. In conclusion findings of this study indicate that ozonation of tertiary effluents of a municipal treatment plant reduces the adverse effects caused by release of mutagens in aquatic ecosystems and does not decrease the viability of bacteria and eukaryotic cells. However, future research is required to find out if, and to which extent these findings can be generalized and which mechanisms account for the detoxification of the wastewater.

Impact of ozonation on ecotoxicity and endocrine activity of tertiary treated wastewater effluent.

Tertiary wastewater treatment plant effluent before and after ozonation (0.6-1.1g O₃/g DOC) was tested for aquatic ecotoxicity in a battery of standardised microbioassays with green algae, daphnids, and zebrafish eggs. In addition, unconjugated estrogen and 17β-hydroxyandrogen immunoreactive substances were quantified by means of enzyme immunoassays, and endocrine effects were analysed in a 21-day fish screening assay with adult male and female medaka (Oryzias latipes). Ozonation decreased estrogen-immunoreactivity by 97.7±1.2% and, to a lesser extent, androgen-immunoreactivity by 56.3±16.5%. None of the short-term exposure ecotoxicity tests revealed any adverse effects of the tertiary effluent, neither before nor after the ozonation step. Similarly in the fish screening assay, reproductive fitness parameters showed no effects attributed to micropollutants, and no detrimental effects of the effluents were observed. Based on the presented screening, ozonation effectively reduced steroid hormone levels in the wastewater treatment plant effluent without increasing the effluents ecotoxicity.

Considerations on methodological challenges for water footprint calculations

We have investigated how different approaches for water footprint (WF) calculations lead to different results, taking sugar beet production and sugar refining as examples. To a large extent, results obtained from any WF calculation are reflective of the method used and the assumptions made. Real irrigation data for 59 European sugar beet growing areas showed inadequate estimation of irrigation water when a widely used simple approach was used. The method resulted in an overestimation of blue water and an underestimation of green water usage. Dependent on the chosen (available) water quality standard, the final grey WF can differ up to a factor of 10 and more. We conclude that further development and standardisation of the WF is needed to reach comparable and reliable results. A special focus should be on standardisation of the grey WF methodology based on receiving water quality standards.

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types.

COST Action ES1403: New and Emerging challenges and opportunities in wastewater REUSe (NEREUS)

Treated urban wastewater is currently widely reused to compensate for dwindling water supplies, as it is considered to be a reliable alternative water source. In addition, the increasing demand for food due to the expanding world population, both in respect to food security and food safety, and therefore for irrigation water, renders wastewater reuse a practice of utmost importance. As a consequence, sustainable and safe urban water cycles are presently of high priority on the policy agendas of many countries around the world. Although reuse has a number of benefits and major advances have been made with respect to producing treated effluents for reuse (e.g., successful removal of metals, reduction of chemical oxygen demand and of other pollution parameters), several important questions are still unanswered and barriers exist regarding the safety/sustainability of reuse practice. Knowledge gaps associated with wastewater reuse include the following: (a) possible elemental interactions that may influence the accumulation of metals/elements in the soil and their subsequent uptake by plants and crops, (b) the fateof organic microcontaminants in receiving environments, and (c) the epidemiological potential of antibiotic resistant bacteria and/or resistance genes (ARB&ARG) released in the environment via treated effluent. Possible implications on food-chain contamination (biomagnification) require much attention, since treated wastewater is not exempt of such contaminants. The effluents’ residual organic matter after conventional treatment consists of a number of recalcitrant organic compounds including potential endocrine disrupting compounds, many types of pharmaceuticals including antibiotics, disinfection by-products, personal care products, metabolites and transformation products, other organic substances (i.e. pesticides, surfactants, biocides, etc.), and not to be forgotten ARB&ARG. In fact, preliminary results suggest that the relative abundance of certain ARG or ARB may even be enriched during the wastewater treatment (Rizzo et al. 2013). This leads to their subsequent release in the terrestrial and aquatic environments through disposal and reuse applications, and the level of risk to environmental and human health is yet to be evaluated. Contamination of the environment, food chain, drinking water, etc with ARB&ARG is presently considered to be a serious public health problem. For this reason, the World Health Organization (WHO) (WHO 2013) characterized the development of AR as one of the major global threats to society and recommends intensive monitoring for the identification/surveillance of critical hot spots (e.g., wastewater treatment plants), aiming at reducing its propagation. In September 2014, a national strategy (The White House 2014) was announced in the USA by the White House that lays out a series of steps to address the decreasing effectiveness of antibiotics, many being similar to those identified by WHO. According to the European Centre for Disease Prevention and Control, it is estimated that infections caused by a subset of ARB are responsible for about 25,000 deaths in Europe annually. In addition, the extra healthcare costs and productivity losses due to ARB are estimated to reach EUR 1.5 billion (European Centre for Disease Prevention and Control 2013). In the USA, equally dramatic numbers are reported by the Centers for Disease Control and Prevention, with AR infections killing at least 23,000 people and sickening 2 million each year (The White House 2014). All these issues have not received significant attention in the framework of the wastewater reuse practice. The EUCOST Action ES1403 (NEREUS) aims at consolidating the existing scattered data related to wastewater reuse and will address the open challenges associated with it. It will provide the platform for a systematic consolidation of data and standardization of methods for assessing emerging hazards associated with wastewater reuse. The Action is chaired by D. Fatta-Kassinos from Nireas-International Water Research Center and Department of Civil and Environmental Engineering of the University of Cyprus and vice-chaired by C.Manaia from Escola Superior de Biotecnologia, Universidade Catolica Portugeusa.

REVIEW ON THE ASSESSMENT OF THE REMOVAL EFFICIENCY OF WASTEWATER TREATMENT PLANTS FOR SELECTED XENOBIOTICS

Wastewater treatment plants (WWTPs) represent a significant source for the input of micro pollutants as endocrine disruptors (EDs) or pharmaceuti- cally active compounds (PhACs) into the aquatic environment. Those xenobiotics show effects to aquatic life at very low concentration e.g. by interfering with their hormone system. As a consequence investigations on the removal potential of various traditional as well as new waste water treatment technologies are undertaken in order to identify appropriate removal of xenobiotics from the urban water cycle. Beside laboratory experiments with synthetic wastewater and addition of a known concentration of the substance of interest, full scale treatment plants representing a broad range of layouts and concepts as well as a broad range of substances analyzed are investigated. In that context removal rates are calculated by comparing inflow and effluent concentration. This paper deals with a review on aspects that have to be kept in mind when assessing the removal potential of full scale wastewater treatment plants in order to avoid systematic errors and to come up with reliable results that can be compared to other plant configurations or operation types. After theoretical considerations an example for a mass balance of a complex waste water treatment plant is given to show a procedure suitable to reveal reliable information on the behavior of xenobiotics during wastewater treatment.

Strain typing of ectomycorrhizal basidiomycetes from subalpine Tyrolean forest areas by random amplified polymorphic DNA analysis

Abstract The application of random amplified polymorphic DNA (RAPD) analysis for the identifcation of ectomycorrhizal symbionts of spruce (Picea abies) belonging to the genera Boletus, Amanita and Lactarius at and below the species level was investigated. Using both fingerprinting [M13, (GTG)5, (GACA)4] as well as random oligonucleotide primers (V1 and V5), a high degree of variability of amplified DNA fragments (band-sharing index 65–80%) was detected between different strains of the same species, hence enabling the identification of individual strains within the same species. The band-sharing index between different species of the same genus (Boletus, Russula and Amanita) was in the range of 20–30%, and similar values were obtained when strains from different taxa were compared. Thus RAPD is too sensitive at this level of relatonship and cannot be used to align an unknown symbiont to a given taxon. We therefore conclude that RAPD is a promising tool for the identification of individual strains, and could thus be used to distinguish indigenous and introduced mycorrhizal strains from the same species in natural ecosystems.